We investigated the hypothesis that methicillin-resistant Staphylococcus aureus (MRSA) isolates developing reduced susceptibilities to daptomycin (DAP; a calcium-dependent molecule acting as a cationic antimicrobial peptide [CAP]) may also coevolve reduced in vitro susceptibilities to host defense cationic antimicrobial peptides (HDPs). Ten isogenic pairs of clinical MRSA DAP-susceptible/DAP-resistant (DAP(s)/DAP(r)) strains were tested against two distinct HDPs differing in structure, mechanism of action, and origin (thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]) and one bacterium-derived CAP, polymyxin B (PMB). Seven of 10 DAP(r) strains had point mutations in the mprF locus (with or without yyc operon mutations), while three DAP(r) strains had neither mutation. Several phenotypic parameters previously associated with DAP(r) were also examined: cell membrane order (fluidity), surface charge, and cell wall thickness profiles. Compared to the 10 DAP(s) parental strains, their respective DAP(r) strains exhibited (i) significantly reduced susceptibility to killing by all three peptides (P < 0.05), (ii) increased cell membrane fluidity, and (iii) significantly thicker cell walls (P < 0.0001). There was no consistent pattern of surface charge profiles distinguishing DAP(s) and DAP(r) strain pairs. Reduced in vitro susceptibility to two HDPs and one bacterium-derived CAP tracked closely with DAP(r) in these 10 recent MRSA clinical isolates. These results suggest that adaptive mechanisms involved in the evolution of DAP(r) also provide MRSA with enhanced survivability against HDPs. Such adaptations appear to correlate with MRSA variations in cell membrane order and cell wall structure. DAP(r) strains with or without mutations in the mprF locus demonstrated significant cross-resistance profiles to these unrelated CAPs.