Daptomycin is one of the last-line antibiotics to treat severe infections caused by methicillin-resistant Staphylococcus aureus. Daptomycin resistance (DAP-R) is emerging and often associated with nonsynonymous mutations in multiple peptide resistance factor (mprF). MprF is a bifunctional enzyme that catalyses the production of a cationic lysylphosphatidylglycerol (L-PG) from an anionic phosphatidylglycerol and translocates L-PG from the inner leaflet to the outer leaflet of the cell membrane. However, how mprF mutations lead to daptomycin resistance is not entirely clear. Here, we utilized resistance mutation sequencing to characterize the full repertoire of mprF mutations within 10,000 DAP-R clones. We identified 24 nonsynonymous mutations in mprF, of which 6 were novel mutations. Introduction of 6 individual mprF mutation into a daptomycin-susceptible S. aureus strain led to DAP-R. Our lipidomic analysis showed that enhanced biosynthesis of L-PG was resulted from amino acid changes in the L-PG synthase domain caused by the mprF mutations. We reconstituted bacterial membranes and directly measure daptomycin-membrane interactions using neutron reflectometry. The actions of daptomycin were compromised on L-PG-rich membranes of the MprF-L826F strain compared with wild-type, and daptomycin accumulated on the cell surface of MprF-L826F. These data suggested that high density of L-PG in membranes trapped daptomycin to prevent membrane penetration and solubilization. This L-PG mediated mechanism is independent of a previously characterized DAP-R mechanism mediated by enhanced cardiolipin biosynthesis. Together, our results illustrate that S. aureus is capable of utilizing diverse metabolic strategies to circumvent antibiotic attack and provide important insights into membrane-targeting therapeutic strategies against this significant pathogen.