ABSTRACTInvasion of host cells is an important feature ofStaphylococcus aureus. The main internalization pathway involves binding of the bacteria to host cells, e.g., endothelial cells, via a fibronectin (Fn) bridge betweenS. aureusFn binding proteins and α5β1-integrin, followed by phagocytosis. The secreted extracellular adherence protein (Eap) has been shown to promote this cellular uptake pathway of not onlyS. aureus, but also of bacteria otherwise poorly taken up by host cells, such asStaphylococcus carnosus. The exact mechanisms are still unknown. Previously, we demonstrated that Eap induces platelet activation by stimulation of the protein disulfide isomerase (PDI), a catalyst of thiol-disulfide exchange reactions. Here, we show that Eap promotes PDI activity on the surface of endothelial cells, and that this contributes critically to Eap-driven staphylococcal invasion. PDI-stimulated β1-integrin activation followed by increased Fn binding to host cells likely accounts for the Eap-enhanced uptake ofS. aureusinto non-professional phagocytes. Additionally, Eap supports the binding ofS. carnosusto Fn-α5β1integrin, thereby allowing its uptake into endothelial cells. To our knowledge, this is the first demonstration that PDI is crucial for the uptake of bacteria into host cells. We describe a hitherto unknown function of Eap—the promotion of an enzymatic activity with subsequent enhancement of bacterial uptake—and thus broaden mechanistic insights into its importance as a driver of bacterial pathogenicity.IMPORTANCEStaphylococcus aureuscan invade and persist in non-professional phagocytes, thereby escaping host defense mechanisms and antibiotic treatment. The intracellular lifestyle ofS. aureuscontributes to the development of infection, e.g., in infective endocarditis or chronic osteomyelitis. The extracellular adherence protein secreted byS. aureuspromotes its own internalization as well as that of bacteria that are otherwise poorly taken up by host cells, such asStaphylococcus carnosus. In our study, we demonstrate that staphylococcal uptake by endothelial cells requires catalytic disulfide exchange activity by the cell-surface protein disulfide isomerase, and that this critical enzymatic function is enhanced by Eap. The therapeutic application of PDI inhibitors has previously been investigated in the context of thrombosis and hypercoagulability. Our results add another intriguing possibility: therapeutically targeting PDI, i.e., as a candidate approach to modulate the initiation and/or course ofS. aureusinfectious diseases.