Vascular endothelium is an exposed target in systemic endovascular Staphylococcus aureus infections. We reported earlier that the proinflammatory and procoagulant activities of primary human umbilical vein endothelial cells (ECs) after binding and ingestion of S. aureus organisms provide the cells effective means for leukocyte-mediated bacterial elimination. Expanding on this, we now show that these ECs exhibit a modest intrinsic capacity for eliminating intracellular S. aureus that was influenced by cytokines relevant to S. aureus infections. Using various EC infection assays, we showed that gamma interferon (IFN-gamma), applied to cultures of ECs prior to or after infection with S. aureus, markedly reduced the level of infection, illustrated by lower percentages of S. aureus-infected ECs and less intracellular bacteria per infected cell. IFN-gamma-activated ECs had unaltered abilities to bind S. aureus and processed ingested bacteria by a seemingly conventional phagocytic pathway. IFN-gamma treatment rescued EC monolayers from severe injury by virulent clinical S. aureus strains or excessive bacterial numbers. Mechanistically, IFN-gamma controls S. aureus infection via IFN-gamma receptor, most likely through stimulation of intrinsic endothelial antibacterial mechanisms but independent of processes that deprive bacteria of intracellular L-tryptophan or iron. The antibacterial activity of IFN-gamma-stimulated ECs coincided with sustained or slightly elevated endothelial proinflammatory responses that supported monocyte recruitment. In conclusion, we identify IFN-gamma as a potent regulatory Th1 cytokine possessing exclusive abilities to augment intrinsic antistaphylococcal effector mechanisms in human ECs without ablating the S. aureus-induced proinflammatory EC responses and, as such, coordinating a protective efficacy of ECs against blood-borne S. aureus infection.