Membrane vesicles (MVs) are nanoscale particles secreted by living bacteria in vitro and in vivo. Bacterial MVs encapsulate various proteins, making them promising candidates for developing vaccines, drug carriers, and cancer immunotherapy agents. However, the mechanisms underlying MV secretion in Gram-positive bacteria remain unclear. Here, we showed that the subinhibitory concentration of oxacillin (OXA) stimulated MV production in Staphylococcus aureus with diverse genetic backgrounds. OXA treatment remarkably increased the expression of sle1, which encodes a main peptidoglycan hydrolase for adjusting peptidoglycan cross-linking. Deletion of sle1 decreased the OXA-mediated MV yield, whereas overexpression of sle1 considerably increased MV production. The accessory regulator SarA increased in response to OXA treatment, and SarA inactivation substantially attenuated OXA-stimulated MV production. We also demonstrated that SarA controlled sle1 expression by directly binding to its promoter region. Thus, the SarA-Sle1 regulatory axis was formed to mediate OXA-induced MV production in S. aureus. MVs derived from OXA-treated S. aureus RN4220 (MVs/OXA) exhibited a smaller particle size compared with those purified from wild-type RN4220; however, proteomic analysis revealed a comparable protein profile between MVs and MVs/OXA. Overall, our research reveals a mechanism underlying OXA-promoted S. aureus MV secretion and highlights the potential application of OXA-induced MVs.