The antibacterial activity and mechanism of silver nanoparticles (Ag-NPs) on Staphylococcus aureus ATCC 6538P were investigated in this study. The experiment results showed the minimum bactericidal concentration (MBC) of Ag-NPs to S. aureus was 20 μg/ml. Moreover, when bacteria cells were exposed to 50 μg/ml Ag-NPs for 6 h, the cell DNA was condensed to a tension state and could have lost their replicating abilities. When S. aureus cells were exposed to 50 μg/ml Ag-NPs for 12 h, the cell wall was breakdown, resulting in the release of the cellular contents into the surrounding environments, and finally became collapsed. And Ag-NPs could reduce the enzymatic activity of respiratory chain dehydrogenase. Furthermore, the proteomic analysis showed that the expression abundance of some proteins was changed in the treated bacterial cell with Ag-NPs, formate acetyltransferase increased 5.3-fold in expression abundance, aerobic glycerol-3-phosphate dehydrogenase decreased 6.5-fold, ABC transporter ATP-binding protein decreased 6.2-fold, and recombinase A protein decreased 4.9-fold.