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The reversal of daptomycin resistance in MRSA to a daptomycin-susceptible phenotype following prolonged passage in selected β-lactams occurs coincident with the accumulation of multiple point mutations in the <i>mprF</i> gene. MprF regulates surface charge by modulating the content and translocation of the positively charged cell membrane phospholipid, lysyl-phosphatidylglycerol (LPG). The precise cell membrane adaptations accompanying such β-lactam-induced <i>mprF</i> perturbations are unknown. This study examined key cell membrane metrics relevant to antimicrobial resistance among three daptomycin-resistant MRSA clinical strains, which became daptomycin-susceptible following prolonged exposure to cloxacillin (‘daptomycin-resensitized’). The causal role of such secondary <i>mprF</i> mutations in mediating daptomycin resensitization was confirmed through allelic exchange strategies. The daptomycin-resensitized strains derived either post-cloxacillin passage or via allelic exchange (vs. their respective daptomycin-resistant strains) showed the following cell membrane changes: (i) enhanced BODIPY-DAP binding; (ii) significant reductions in LPG content, accompanied by significant increases in phosphatidylglycerol content (<i>p</i> < 0.05); (iii) no significant changes in positive cell surface charge; (iv) decreased cell membrane fluidity (<i>p</i> < 0.05); (v) enhanced carotenoid content (<i>p</i> < 0.05); and (vi) lower branched chain fatty acid profiles (antiso- vs. iso-), resulting in increases in saturated fatty acid composition (<i>p</i> < 0.05). Overall, the cell membrane characteristics of the daptomycin-resensitized strains resembled those of parental daptomycin-susceptible strains. Daptomycin resensitization with selected β-lactams results in both definable genetic changes (i.e., <i>mprF</i> mutations) and a number of key cell membrane phenotype modifications, which likely facilitate daptomycin activity.