The escalating prevalence of methicillin-resistant Staphylococcus aureus (MRSA) underscores the critical necessity for innovative antibacterial strategies. Berberine sulfate, a naturally occurring antimicrobial agent, demonstrates inhibitory effects against MRSA; however, its precise molecular targets and mechanisms of action remain inadequately defined. This study sought to systematically elucidate the regulatory mechanisms of berberine sulfate on MRSA, with particular emphasis on its influence on protein expression and post-translational modifications. Through quantitative proteomic analysis, 650 differentially expressed proteins were identified. Subsequent acetylome profiling revealed 38 differentially acetylated sites, among which berberine sulfate specifically induced K82 acetylation of the global regulator sarA. Functional analyses further revealed that the K82 mutant sarA exhibited impaired binding affinity to the agr promoter. These findings suggest a comprehensive dynamic response and potential molecular mechanism mediated by anti-MRSA effect of berberine sulfate.