Mary Winchester, (depicted by Amy Gumenick in the time-travel episodes "In the Beginning" and "The Song Remains the Same"), is the wife of John Winchester and mother of Sam and Dean. Her parents, Samuel and Deanna Campbell, were hunters, and she was raised into a life of hunting. Dean, sent back in time by the angel Castiel, unknowingly brings Mary to Azazel's attention through his actions. Azazel kills her parents and then-boyfriend John Winchester (a non-hunter unaware of Mary's hunter lifestyle), after which he bargains with Mary for John's life, offering to resurrect John if she allows him to enter her house ten years later. Not knowing Azazel's intentions, she agrees. Eventually marrying John and leaving the life of a hunter, she gives birth to Dean and Sam.

In the pilot episode, it is revealed, six months after Sam's birth, Mary was awakened by sounds of him crying in his crib. She discovered Azazel there - later revealed to have been feeding Sam his demonic blood - and confronted him, but was pinned to the ceiling by him and slashed across her abdomen, eventually bursting into flames.
In the episode Home, her sons return to their childhood home in Lawrence, Kansas to investigate a recurring dream Sam experiences. Mary's spirit seems to haunt the house, and a poltergeist is in the house with her. After the poltergeist pins Sam to the wall, Mary appears before Sam as a fiery figure and tells him she is sorry. Her spirit then fights with and neutralizes the poltergeist, forcing both spirits out of the home.
In The Kids are Alright, it is revealed all of Mary's friends and acquaintances have been killed off in the years since her death, although the reasons for this are never explained.