The development of antimicrobials that exert therapeutic efficacy by novel mechanisms is crucial toward combatting multidrug-resistant bacteria. We previously reported a novel antibiotic, lysocin E, that is effective against methicillin-resistant Staphylococcus aureus (MRSA)1. Lysocin E exhibits more prominent therapeutic effects against S. aureus in a mouse systemic infection model compared with its basal antibacterial activity in vitro, but the detailed mechanism underlying this discrepancy was unclear. Here we show that serum or plasma from various species, including humans, increases the antimicrobial activity of lysocin E, and identified apolipoprotein A-I (ApoA-I) as an enhancing factor in the serum. Using gene knockout mice, we further revealed that ApoA-I contributes to the therapeutic effects of lysocin E. The binding capacity of lysocin E to ApoA-I was enhanced by lipid II, an intermediate component of S. aureus cell wall synthesis in the membrane, and ApoA-I enhanced the membrane-damaging activity of lysocin E at a sub-minimum inhibitory concentration (sub-MIC). Our results are the first to demonstrate that antimicrobial activity can be potentiated through interactions of host serum proteins with microbial components to enhance the therapeutic effect, which broadens the strategies for developing antimicrobials by taking advantage of host-microbe interactions.