Integrin ␣31 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of ␣31 can affect angiogenesis either positively or negatively, either a direct in vivo role for ␣31 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of ␣3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where ␣3 is deleted specifically in endothelial cells (ec-␣3؊/؊). Here we show that ec-␣3؊/؊ mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that ␣31 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial ␣31 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.