Silver ions (Ag⁺) have attracted profound attention due to their broad-spectrum antimicrobial activities. Although the antibacterial properties of silver have been well known for many centuries, its mechanism of action is not fully understood and its protein targets remain largely unknown. Staphylococcus aureus (S. aureus) is the leading cause of hospital- and community-acquired infections. Staphylococcal accessory regulator protein family from Staphylococcus aureus has been found to play vital roles in the regulation of virulence genes. In this study, we demonstrated that silver ions bind to the staphylococcal accessory regulator A (SarA) of S. aureus via its cysteine residues. Importantly, binding of silver ions leads to functional disruption of SarA. In addition, qRT-PCR experiments showed that silver also significantly attenuated the mRNA transcription levels of the genes which SarA regulated. Overall, these results provide new insights into the antibacterial mechanism of silver ions.