We previously reported that the FAS1 domains of betaig-h3 bear motifs that mediate endothelial cell adhesion and migration via interactions with alphavbeta3 integrin and regulate angiogenesis. In the present study, we show that the fourth FAS1 domain, designated fastatin, inhibits endothelial adhesion and migration, not only to betaig-h3, but also fibronectin and vitronectin, in a RGD-dependent manner. Fastatin and other FAS1 domains suppress endothelial cell tube formation and in vivo neovascularization in a Matrigel plug assay. The antiangiogenic activity of fastatin is associated with antitumor activity in mouse tumor models. Fastatin additionally induces apoptosis in several cells expressing alphavbeta3 integrin, including endothelial cells. Binding of fastatin to alphavbeta3 integrin inhibits phosphorylation of focal adhesion kinase, Raf, extracellular signal-regulated kinase, Akt, and mammalian target of rapamycin. Fastatin is thus the first endogenous angiogenesis regulator identified that inhibits both endothelial cell migration and growth by binding to alphavbeta3 integrin. Our data suggest that FAS1 domains from all possible forms of the four human FAS1 family proteins are potential endogenous regulators for pathologic angiogenesis. Moreover, FAS1 domains such as fastatin may be developed into drugs for blocking tumor angiogenesis.