Toxin-antitoxin (TA) systems are ubiquitously found in bacteria and are related to cell maintenance and survival under environmental stresses such as heat shock, nutrient starvation, and antibiotic treatment. Here, we report for the first time the crystal structure of the Staphylococcus aureus TA complex YoeB(Sa1)-YefM(Sa1) at a resolution of 1.7 angstrom. This structure reveals a heterotetramer with a 2:2 stoichiometry between YoeB(Sa1) and YefM(Sa1). The N-terminal regions of the YefM(Sa1) antitoxin form a homodimer characteristic of a hydrophobic core, and the C-terminal extended region of each YefM(Sa1) protomer makes contact with each YoeB(Sa1) monomer. The binding stoichiometry of YoeB(Sa1) and YefM(Sa1) is different from that of YoeB and YefM of E. coli (YoeB(Ec) and YefM(Ec)), which is the only structural homologue among YoeB-YefM families; however, the structures of individual YoeB(Sa1) and YefM(Sa1) subunits in the complex are highly similar to the corresponding structures in E. coli. In addition, docking simulation with a minimal RNA substrate provides structural insight into the guanosine specificity of YoeB(Sa1) for cleavage in the active site, which is distinct from the specificity of YoeB(Ec) for adenosine rather than guanosine. Given the previous finding that YoeB(Sa1) exhibits fatal toxicity without inducing persister cells, the structure of the YoeB(Sa1)-YefM(Sa1) complex will contribute to the design of a new category of anti-staphylococcal agents that disrupt the YoeB(Sa1)-YefM(Sa1) complex and increase YoeB(Sa1) toxicity. (c) 2020 Elsevier Inc. All rights reserved.