Apolipoprotein A-I (apoA-I), the main protein component of high density lipoprotein (HDL), is well recognized for its antiatherogenic, antioxidant, and antiinflammatory properties. Here, we report a novel role for apoA-I as a host defense molecule that contributes to the complement-mediated killing of an important gastrointestinal pathogen, Gram-negative bacterium Yersinia enterocolitica. We specifically show that the C-terminal domain of apoA-I is the effector site providing the bactericidal activity. Although the presence of the lipopolysaccharide O-antigen on the bacterial surface is absolutely required for apoA-I to kill the bacteria, apoA-I does not interact with the bacteria directly. To the contrary, exposure of the bacteria by serum proteins triggers apoA-I deposition on the bacterial surface. As our data show that both purified lipid-free and HDL-associated apoA-I displays anti-bacterial potential, apoA-I mimetic peptides may be a promising therapeutic agent for the treatment of certain Gram-negative infections.