Splicers, also known as Aggressors, are the main enemies that Jack encounters over the course of the game. The remains of Rapture's human population, they are the result of a planned attack by Atlas (Fontaine) on New Year's Day, 1959 to end the civil war between him and Ryan. They apparently murdered the sane population. Many of them still wear Masquerade Ball masks, and Atlas asks, "I wonder why they wear masks? Maybe it's because they have a shred of humanity left, and they're ashamed of what they've become." Apparently due to the biological weapons attack, they have become biologically dependent on ADAM to survive. Wandering the corridors and the tunnels of Rapture, they search for ADAM, usually in the form of Little Sisters, which often puts them at odds with their Big Daddy protectors-something that Jack can easily exploit to help him survive.

Splicers tend to wear very little armor, if any, but they have advanced physical strength and tend to be extremely aggressive in contrast to the calmer Big Daddys. They will sometimes work in groups, use plasmids and sometimes even play dead to get the advantage on the player.