The secretome of the Staphylococcus aureus includes specific pathway-exported proteins, as well as cytoplasmic proteins with unknown mechanisms of secretion. As one component of the S. aureus secretome, extracellular membrane vesicles (MVs) are also comprised of secreted and cytoplasmic proteins. However, the contribution of MVs to the secretome of S. aureus has not been investigated. To address this, we performed a proteomic analysis of MVs purified from S. aureus strain MRSA252 along with a similar analysis of the culture supernatans before (S+MV) and after (S-MV) depletion of MVs.