Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with α_Vβ₃, in Mn²⁺‐treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn²⁺ resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and α_Vβ₃, and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI–α_Vβ₃ complex, which dissociates upon reduction. In addition, PDI in a complex with α_Vβ₃ induces conversion of the integrin to the ligand‐competent high‐affinity state, as evidenced by increased binding of vitronectin. The membrane‐impermeable sulfhydryl blockers 3‐N‐maleimidylpropionyl biocytin 3‐N‐maleimidylpropionyl biocytin and p‐chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn²⁺. Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI–α_Vβ₃ stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn²⁺, probably mediated by disulfide bonds. Thus, we show that Mn²⁺ simultaneously modulates the thiol isomerase activity of PDI that is bound to α_Vβ₃ and induces its transition to the ligand‐competent state, suggesting an alternative mechanism of integrin regulation.