Dean Winchester - He is the eldest son of John and Mary, the older brother of Sam, and the older paternal half-brother of Adam. He is one of the three surviving members of the Winchester family. When he was young he was forced to grow up right away in order to take care of his little brother, Sam, which eventually became who he was in life. He was raised into hunting by his father, John. He was recently killed by the angel Metatron and resurrected as a Knight Of Hell. He has since been cured by his brother Sam.
Sam Winchester - He is the youngest son of John and Mary. He is the younger brother of Dean and the older paternal half-brother of Adam. He is one of the three surviving members of the Winchester family. His father, John, hadn't brought him into hunting right away like his older brother, Dean. Instead he had to find out who his family was the hard way and eventually was forced to go into "The Family Business". He had always been the odd one out, but eventually accepted the fact that hunting was who he is.
John Winchester (deceased) - Head of the Winchester family. He was the husband of Mary. He had two sons with Mary, Dean and Sam. He had no knowledge of the supernatural world before his wife's murder, but he became a hunter to avenge Mary. Sometime after his wife's murder, he had another son out of wedlock, Adam. He traded his life along with the Colt so Azazel would heal and resurrect Dean after a fatal car crash. Later, he escaped Hell while the Devil's Gate was open and helped his sons to defeat Azazel. After that, he disappeared in a flash of light.
Mary Winchester (née Campbell) - She is the wife of John and the mother of Dean and Sam. She is the matriarch of the Winchester family. She named her two sons after her deceased parents, Samuel and Deanna. Her parents raised her as a hunter, but she gave up hunting after both her parents were killed by Azazel. She was killed by Azazel when she went into Sam's room while Azazel was feeding Sam his blood. She never told John about the supernatural world or her former life as a hunter. Dean and Sam didn't learn about her hunter origins until Castiel sent Dean back in time. She was resurrected by The Darkness. She is currently trapped in an Alternate Reality after being sent there with Lucifer.
Cain and Abel (both deceased) - It was revealed by the Archangel Michael that through the Winchester bloodline, Dean, Sam, Adam and John are descendants of Cain and Abel, making John a vessel and Dean his true vessel. In The Executioner's Song, the now-demonic Cain was killed by Dean
Henry Winchester (deceased) - He was the father of John Winchester, the father-in-law of Mary Campbell and the grandfather of Dean, Sam and Adam. He was entering his final initiation as a Man of Letters in 1958, when they were attacked by the powerful demon Abaddon. Through a spell, he managed to flee to 2013, there he met his grandsons. He teamed up with them to stop Abaddon but died in the process.
Emma (deceased) - Dean's daughter with an Amazon, Lydia. She was killed by Sam while she was attempting to carry out the ritualistic killing of her father.
Adam Milligan (trapped in Lucifer's cage) - He is the illegitimate son of John with nurse Kate Milligan. Unlike his older half-brothers, Sam and Dean, John did not tell him about the supernatural world. He and his mother were killed by ghouls. He was brought back to life by the angels under the impression that he would be the vessel of Michael and would get his mother back, but he was actually bait to get Dean to say "Yes" to Michael. When this plan failed, Michael gave up on Dean and decided to use Adam as a vessel. He is currently still in Lucifer's Cage with Michael.
Millie Winchester (unknown)-She is Henry's wife and John's mother. Never appeared in the series.
Bobby Singer (deceased) - A fellow hunter, who helped the Winchester brothers with many of their cases and sometimes sent them on hunts. The brothers see Bobby as a surrogate father. When they were children, Sam and Dean would call him "Uncle Bobby". Bobby was shot and killed by the leviathan Dick Roman when he, Sam, and Dean were trying to defeat the Leviathans. He remained as a ghost to help Sam and Dean, but he asked to be banished to avoid becoming a vengeful spirit. After being banished, Bobby's soul was collected by a rogue reaper and sent to Hell. Sam rescued his soul from Hell and delivered him to Heaven.
Castiel - The angel that saved Dean from Hell, he started out as an emotionless ally who the Winchesters, especially Dean, were originally more annoyed with than grateful. Over time, Castiel became one of their greatest friends and allies. He rebelled against Heaven and the other angels for the Winchesters. After the Apocalypse was stopped, he raised Sam from Lucifer's cage and began a civil war in Heaven against Raphael. To win, he secretly teamed up with Crowley to absorb Purgatory's souls, lying to and betraying the Winchesters. Feeling regret, he decided to repent for the devastation he caused in Heaven and on Earth. He was later stabbed and killed by Lucifer. Castiel was sent to The Empty where he was to sleep for eternity but awoke because of Jack and met the Cosmic Entity whose slumber he disturbed, to ensure both get what they want Castiel demanded that he be released from the realm and as a result he was resurrected. Castiel reunited with Sam and Dean but was captured along with Lucifer by Asmodeus, the Princes of Hell. He escaped and warned the Winchesters of an impending threat to their world.
Ellen Harvelle (deceased) - A fellow hunter, who was a friend of John Winchester. Like Bobby, she gave guidance to Sam and Dean about hunting. She was like a mother figure to the two brothers, on one occasion lecturing them about not calling her enough to inform her that they were all right. She died trying to help Dean and Sam kill Lucifer with the Colt by blowing herself up to kill hellhounds.
Jo Harvelle (deceased) - A fellow hunter, who was the daughter of Ellen and friend to Dean and Sam. She held romantic feelings for Dean, while he considered her as a sister. She died trying to help Dean and Sam kill Lucifer with the Colt by getting mortally wounded by a hellhound.
Ash (deceased) - A friend to Dean and Sam. Although not a hunter, he attempted to help the brothers track down Azazel. He died in a fire along with other hunters. He now happily resides in Heaven.
Pamela Barnes (deceased) - A psychic, who was a friend of Bobby Singer. She lost her eyes trying to figure out what being rescued Dean from Hell, bringing him back to life, but she only learned the being's name, Castiel. She also helped Anna regain her memories of being an angel. She helped by ripping Sam and Dean's souls from their bodies, but she lost her life protecting the brothers' bodies from demons. She is now living happily in Heaven.
God - God went to Earth disguised as a prophet named Chuck Shurley and wrote several Supernatural novels. After the Winchester brothers found out about him, they confronted him. Afterward, Chuck helped them on several occasions. In Season 11, he revealed himself to the brothers as God and promised to help them against the Darkness.
Jody Mills - She is the sheriff in Sioux Falls, where Bobby lived. The Winchesters saved her life and opened her eyes to the other side. In return, she helped them and Bobby. She called for Bobby's help from Leviathans and inadvertently helped Bobby discover their weakness to Borax. Later on, she too becomes a mother figure to Sam and Dean, inviting them to family dinners and threatening them with her 'mom voice'. She later helps the Winchesters with their hunts.
Garth Fitzgerald IV - Formerly a laid-back hunter, who the brothers have teamed up with on three separate occasions. While on a werewolf hunt, he was bitten. When he tried to commit suicide, he's found by another werewolf, Bes, who he later married. Because they only feed on animals, he decided to live among them and is now a member of their pack.
Charlie Bradbury (deceased) - A hacker who worked for Richard Roman. She helped Sam and Dean hack into Dick Roman's email and deleted their personal information from Frank's hard drive. Over a year later, they crossed paths with her again. They trained her to be a hunter, and she became like a sister to them. She was killed by Eldon Frankenstein in the episode "Dark Dynasty".
Kevin Tran (deceased) - A young prophet who met the Winchesters when he tried to take the Leviathan tablet from them. He was captured by Crowley in "Survival of the Fittest", but later managed to escape and spent a year on the run until the brothers found him again. Kevin helps the Winchesters by deciphering the demon tablet that tells how to keep demons out of Earth forever. In season 8, he is again caught by Crowley who tricks him with fake demons playing Dean and Sam. But he eventually figures out that they're demon actors and escapes them too, and was MIA for a time. Kevin was smote by Gadreel (whilst possessing Sam), and killed. Dean gave him a hunter's funeral. Later Kevin appears in "Captives" as a ghost looking for his mother. Kevin's ghost returns once again in All in the Family to confirm God's identity before God sends his soul to Heaven.
Crowley (deceased) - The demon Crowley and the Winchesters have never had a great relationship and have been vicious enemies with each other in the past, but have multiple times put aside their differences to fight for the greater good or defeat a common enemy. Crowley first meets and helps the Winchesters, Castiel and Bobby Singer in preventing the Apocalypse. in season 6, Crowley and the brothers are enemies due to Crowley teaming up with Castiel to secretly open Purgatory, In season 7, Crowley is again allied with the brothers to defeat Dick Roman and the Leviathans. the brothers and Crowley in season 8 are enemies again due to the Winchesters wanting to close the gates of Hell, and in season 9 and 10, Crowley and the brothers are allies once more, with Dean and Crowley even developing a bond with each other. Season 11 sees the brothers and Crowley working with each other at least once, in a failed attempt to speak with Lucifer and not let the archangel escape Hell. He later sacrificed himself for the Winchesters in an attempt to seal Lucifer in an alternate reality.
Lucifer (during All in the Family and We Happy Few): Tabling his problems with the Winchesters, Lucifer agreed to help them defeat the Darkness, after she brutally tortured him to the point where the Archangel couldn't even teleport. The alliance strengthened after Lucifer finally forgave God. It broke after God departed from Earth, leaving Lucifer with a sense of abandonment again.
The Darkness (after season 11): Reconciling with God, the Darkness dropped any enmity she had against the Winchesters, and even resurrected Mary as a thank-you gift for Dean.
Jack (Season 13): The son of Lucifer and Kelly Kline, who was born following the defeat of Lucifer. He is taken in by the brothers since he had nowhere to go and tries his best to be an ally to the brothers. His lack of control of his powers has caused Sam to teach him due to his understanding of it while Dean did not trust him, even swearing to kill him should he turn on them until Jack saved him from a shape shifter. Jack would later call the Winchesters his family which Dean and Sam agreed with.
Claire Novak - Claire is the daughter of Jimmy Novak, the vessel of Castiel. She was used by Castiel for a short amount of time before his father demanded his self-sacrifice. Claire ends up at a foster home, escaping repeatedly to find her mother and bumping into the Winchesters and Castiel again in the process. When her mother is killed by Tamiel, she urges to become a hunter. She now lives together with Jody Mills and Alex, a hunter in her own right.
The name Winchester may be a pun on the rifle of the same name. In the episode, "The Benders", this is referenced.
The season 8 episode, "As Time Goes By" marked the first time a Winchester outside of the brothers and John appeared. Previously, only the Campbells' extended family was shown.
Though John grew up without a father, it should be noted that in "In The Beginning", in 1973 a guy at a diner says to a young John Winchester "say hello to your old man for me", implying the presence of a paternal figure.
We learn in "Mother's Little Helper" that Henry was doing his final initiation and being out in the field scared him as he feared if something were to happen to him it would leave his wife a widow and his son fatherless. John later also states that he is a "mechanic from a family of mechanics", but never specifically said his father was a mechanic.