The mazEF homologs of Staphylococcus aureus, designated mazEF(sa), have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF(Sa), as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF(Sa) leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF(Sa) is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF(Sa) cleaves single-strand RNA preferentially at the 5' side of the first U or 3' side of the second U residue within the consensus sequences VUUV' (where V and V' are A, C, or G and may or may not be identical). Binding studies confirmed that the antitoxin MazE(Sa) binds MazF(Sa) to form a complex to inhibit the endoribonuclease activity of MazF(Sa). Contrary to the system in E. coli, exposure to selected antibiotics augmented mazEF(sa) transcription, akin to what one would anticipate from the environmental stress response of the sigB system. These data indicate that the mazEF system of S. aureus differs from the gram-negative counterparts with respect to mRNA cleavage specificity and antibiotic stresses.