[Objective] To study the effect of genistein on methicillin-resistant Staphylococcus aureus (MRSA) efflux proteins. [Methods] Combined drug sensitivity test was used to detect the effect of genistein on the sensitivity of MRSA to ciprofloxacin. The isobaric tags for relative and absolute quantitation (iTRAQ) technology were used to detect the change of bacterial cell protein expression after genistein was applied to MRSA41577. The bioinformatics method was used to analyze the significantly different proteins. qPCR and Nile Red efflux assay were used to explore the mechanism of drug resistance-related proteins mediating bacterial resistance. [Results] Combined drug sensitivity test showed that genistein enhanced the sensitivity of MRSA to ciprofloxacin. A total of 129 significantly different proteins were detected by iTRAQ technology, including 60 proteins with up-regulated expression and 69 proteins with down-regulated expression. Bioinformatics analysis showed that there were about 14 proteins related to bacterial resistance, of which PstB, PstS and other proteins mainly mediated bacterial resistance through the active efflux system. qPCR results showed that compared with the control group, PstB and PstS gene expression decreased by 51.6% and 78.6%, respectively. Nile Red efflux assay showed there was a competitive relationship between genistein and Nile Red, so that genistein was a competitive inhibitor of MRSA41577. [Conclusion] Genistein reverses bacterial resistance by reducing mRNA expression of the efflux genes pstB and pstS, and affected the expression of efflux proteins PstB and PstS of MRSA41577. Besides, genistein is also a competitive efflux inhibitor of MRSA41577 and plays antibacterial effect by competing with substrates for efflux, which makes antibacterial drugs stay in the bacteria.