Functional cooperation between integrins and growth factor receptors has been reported for several systems, one of which is the modulation of insulin signaling by αvβ3 integrin. Plasminogen activator inhibitor type‐1 (PAI‐1), competes with αvβ3 integrin for vitronectin (VN) binding. Here we report that PAI‐1, in a VN‐dependent manner, prevents the cooperation of αvβ3 integrin with insulin signaling in NIH3T3 fibroblasts, resulting in a decrease in insulin‐induced protein kinase B (PKB) phosphorylation, vascular endothelial growth factor (VEGF) expression and cell migration. Insulin‐induced HUVEC migration and angiotube formation was also enhanced in the presence of VN and this enhancement is inhibited by PAI‐1. By using specific PAI‐1 mutants with either VN binding or plasminogen activator (PA) inhibiting activities ablated, we have shown that the PAI‐1‐mediated interference with insulin signaling occurs through its direct interaction with VN, and not through its PA neutralizing activity. Moreover, using cells deficient for uPA receptor (uPAR) we have demonstrated that the inhibition of PAI‐1 on insulin signaling is independent of uPAR‐VN binding. These results constitute the first demonstration of the interaction of PAI‐1 with the insulin response.