Antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms. Here we demonstrate that the outer membrane lipoprotein, Lpp, of Enterobacteriaceae interacts with and promotes susceptibility to the bactericidal activities of AMPs. The oligomeric Lpp was specifically recognized by several cationic α-helical AMPs, including SMAP-29, CAP-18, and LL-37; AMP-mediated bactericidal activities were blocked by anti-Lpp antibody blocking. Blebbing of the outer membrane and increase in membrane permeability occurred in association with the coordinate internalization of Lpp and AMP. Interestingly, the specific binding of AMP to Lpp was resistant to divalent cations and salts, which were able to inhibit the bactericidal activities of some AMPs. Furthermore, using His-tagged Lpp as a ligand, we retrieved several characterized AMPs, including SMAP-29 and hRNase 7, from a peptide library containing crude mammalian cell lysates. Overall, this study explores a new mechanism and target of antimicrobial activity and provides a novel method for screening of antimicrobials for use against drug-resistant bacteria.