Cationic antimicrobial peptides (CAMPs) such as defensins and cathelicidins are an integral part of the human innate immune system. They protect mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophil granulocytes. Staphylococcus aureus, a major human pathogen, achieves resistance to human defense peptides by modifying anionic phosphatidylglycerol with positively charged L-lysine, resulting in repulsion of CAMPs. This lysinylation is mediated by the novel S. aureus virulence factor MprF. Inactivation of mprF, which has no similarity with genes of known function but is found in the genomes of many bacterial pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa and Enterococcus faecalis, results in the loss of lysylphosphatidylglycerol (L-PG), increased inactivation by CAMP-producing neutrophils, and attenuated virulence. Furthermore, it has recently been shown that mutations in mprF lead to resistance of S. aureus to the novel lipopeptide antibiotic daptomycin. The production of L-PG is dependent on phosphatidylglycerol and lysyl-tRNA but the precise mechanism of L-PG biosynthesis remained unknown. Here we characterize the L-PG synthase domain of MprF and demonstrate that MprF represents a new class of enzyme with a complex structure. A detailed analysis of the mechanism of L-PG synthesis may help to find inhibitors of MprF, which would render a great number of bacterial pathogens susceptible to human host defenses and could help to combat multiple resistant, L-PG producing bacteria, such as S. aureus, Pseudomonas aeruginosa and Enterococcus faecalis.