John Winchester turned to hunting supernatural threats after a demon killed his wife. He dedicated his life to this pursuit and rigorously trained his sons, Sam and Dean, from childhood. His instruction included judo, karate, and marksmanship, preparing them for constant danger. His later disappearance spurred Sam and Dean to search for him. During this time, he communicated indirectly, like through psychic Missouri Mosley, avoiding direct contact as he believed reunion premature. He investigated cases independently, such as cattle mutilations linked to vampires, while his sons tracked similar leads.

The family reunited to hunt the Yellow-Eyed Demon, using the powerful Colt weapon. After Dean suffered life-threatening injuries in a car crash, John made a demonic deal: trading his soul and the Colt to save Dean's life. When the Devil's Gate opened, releasing Hell's spirits, John's soul escaped. He physically intervened in a confrontation, distracting the Yellow-Eyed Demon and causing it to drop the Colt, enabling Dean to kill it. John shared a tearful farewell with his sons before vanishing. This version consistently wore a hat, differing from his live-action portrayal, and his soul's escape from Hell possessed greater ambiguity regarding the exact mechanism.