Integrins, transmembrane glycoprotein receptors, play vital roles in pathological angiogenesis, but their precise regulatory functions are not completely understood and remain controversial. This study aims to assess the regulatory functions of individual beta subunits of endothelial integrins in angiogenic responses induced by vascular endothelial growth factor (VEGF). Inhibition of expression of β1, β3, or β5 integrins in endothelial cells resulted in down regulation of EC adhesion and migration on the primary ligand for the corresponding integrin receptor, while no effects on the recognition of other ligands were detected. Although inhibition of expression of each subunit substantially affected capillary growth stimulated by VEGF, the loss of β3 integrin was the most inhibitory. EC stimulation by VEGF induced formation of the high affinity (activated) state of αVβ3 in a monolayer and activated αVβ3 was co-localized with VEGF receptor-2 (VEGFR-2). Inhibition of expression of β1, β3, or β5 did not affect expression levels of VEGFR-2 in EC. However, inhibition of β3, but not β1 or β5, resulted in substantial inhibition of VEGFR-2 phosphorylation stimulated by VEGF. Exogenous stimulation of αVβ3 integrin with activating antibodies augmented VEGF-dependent phosphorylation of VEGFR-2, whereas integrin blockade suppressed this response. Most importantly, activated αVβ3 was detected on endothelial cells of tumor vasculature. Activation of αVβ3 was substantially increased in highly-vascularized tumors as compared to normal tissues. Moreover, activated αVβ3 was co-localized with VEGFR-2 on endothelial cells of proliferating blood vessels. Together, these results show the unique role of αVβ3 integrin in cross-talk with VEGFR-2 in the context of pathological angiogenesis.