In Space Marine, the player engages enemies with both melee and ranged from a third-person point of view. The player character, Titus, has regenerative abilities. When his health gets low, performing some actions can allow Titus to regain a portion of his health. Titus is accompanied by the characters Sidonus and Leandros for most of the missions, though some missions are performed alone. The game features a fury meter that, when filled up, allows the player to either slow time for aiming or perform powerful melee attacks.

The starting weapons include a combat knife and a pistol, though the player can acquire other weapons. The game also includes a new weapon not featured in the table-top game known as the Vengeance Launcher, which can shoot sticky grenades.

The game's main antagonists are the Orks and the Forces of Chaos. The Orks are an alien race of brutish warriors. The Forces of Chaos are an even greater threat, and can summon monsters that live in a parallel dimension known as The Warp. Portals conjured by the Traitor Legions allow daemons to enter the battlefield.