We have previously shown that angiomotin (Amot) mediates angiostatin inhibition of endothelial migration and tube formation in vitro. A crucial role of angiomotin in regulating endothelial cell motility is indicated by the findings that knockdown of Amot in zebrafish reduces the number of filopodia of endothelial tip cells and severely impairs the migration of intersegmental vessels. In addition, targeting angiomotin using DNA vaccination inhibits angiogenesis and tumor growth in vivo. In this report, we have generated antibodies that, similar to angiostatin, bind to angiomotin on the endothelial cell surface. These antibodies inhibited FGF-2 and vascular endothelial growth factor (VEGF) -induced endothelial migration in the Boyden chamber assay. Furthermore, the anti-Amot B06 antibody significantly reduced the number of endothelial filopodia and inhibited vessel migration during retinal angiogenesis in vivo. We also show that systemic or local treatment with this antibody inhibits pathological blood vessel formation associated with tumor growth or laser-induced choroid neovascularization of the eye. These findings provide a rationale for using angiomotin antibodies for specifically targeting endothelial migration in angiogenesis-dependent diseases.