To investigate the mechanism of azalomycin F5a against methicillin-resistant Staphylococcus aureus (MRSA), the conductivity of MRSA suspension and the adenylate kinase activity of MRSA culture were determined with the intervention of azalomycin F5a, which were significantly increased compared to those of blank controls. This inferred that azalomycin F5a could lead to the leakage of cellular substances possibly by increasing permeability to kill MRSA. As phospholipid bilayer was mainly responsible for cell-membrane permeability, the interaction between azalomycin F5a and cell-membrane lipids was further researched by determining the anti-MRSA activities of azalomycin F5a combined with cell-membrane lipids extracted from test MRSA or with 1,2-dipalmitoyl-sn-glycero-3-phospho-glycerol (DPPG) for possible molecular targets lying in MRSA cell-membrane. The results indicated that the anti-MRSA activity of azalomycin F5a remarkably decreased when it combined with membrane lipids or DPPG. This indicated that cell-membrane lipids especially DPPG might be important targets of azalomycin F5a against MRSA.