Mary Winchester (born 1954) is the wife of John Winchester, and mother of Dean and Sam. She gave birth to Dean on January 24, 1979 and Sam on May 2, 1983.

In the episode "Pilot," it is revealed that on November 2, 1983, Mary woke after putting her six-month-old son, Sam, to bed. She entered his nursery to find someone she thought to be John leaning over his crib. Returning to bed, she noticed a television flickering downstairs. She found her husband John asleep in the living room, and ran back to Sam's nursery. Hearing his wife's screams, John ran into the nursery. In Sam's room, everything appeared normal, until he saw droplets of blood next to Sam's head. Looking up, he found Mary pinned to the ceiling with a large gash through her stomach. She then burst into flames, dying at the age of 29. She was murdered by "The Demon," the series' villain.

In 2005, her sons returned to their childhood home to investigate a recurring dream Sam had experienced in the episode "Home." Mary's spirit seemed to haunt the house in Lawrence, Kansas, and a poltergeist was in the house with her, though the psychic Missouri Moseley never felt their presence. After the poltergeist pinned Sam to the wall, Mary appeared as a fiery figure. When Dean broke through the locked door to reach him, Mary told Sam that she was sorry. Turning to the ceiling, she told the poltergeist to leave her sons alone. Her spirit fought with and canceled out the poltergeist, forcing both spirits out of the home.

In All Hell Breaks Loose, Part 1, the Demon shows Sam the night Mary was killed because she was in the wrong place at the wrong time. When Mary ran back into the nursery she said "It's You!" to the Demon. It remains unclear how she could have known the Demon. In Supernatural: Origins, it seems that Mary has a sister that the Demon was apparently after, but this is unconfirmed.

In The Kids are Alright we learn that all of Mary's friends and acquaintences have been killed off over the years since her death.