This season focuses on Sam and Dean continuing to hunt demons, the introduction of mysteriously benevolent demon Ruby (Cassidy), and a rivalry with Bela Talbot(Cohan), a professional thief who steals the Colt. Much is made of the fact that Dean's crossroad deal means that this is his last year on Earth before going to Hell.Lilith serves as a recurring villain throughout the season.

Supernatural's season three, along with many other American television shows, was caught in the middle of a contract dispute between the Writers Guild of America, East (WGAE), Writers Guild of America, west (WGAw) and the Alliance of Motion Picture and Television Producers (AMPTP). The dispute led to a strike by the writers, which caused this season to end prematurely with only sixteen episodes being produced, instead of the standard twenty-two episodes.