We examined the function of α₄β₁ integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. α₄β₁ supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by α₄β₁, whereas antagonism of fibroblast growth factor-2–stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an α₄β₁-dependent manner. Soluble α₄β₁ antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1–dependent and not observed in explants from thrombospondin-1^−/− mice. Antagonizing α₄β₁ may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the α₄β₁ binding sequence stimulate angiogenesis in vivo. Therefore, α₄β₁ is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.