The game follows an elite group of assassins called the "killer7". The assassins, physical manifestations of a man named Harman Smith, perform hits on behalf of the United States government. Through these missions, the killer7 uncover a deeper conspiracy regarding the role of Japan in US politics and secrets about the nature of their organization.

Killer7 features first-person shooter elements and a unique on rails control scheme, but the core adventure-style gameplay has been compared to Myst and Snatcher.