Angiogenesis is essential for tumor growth and metastasis. Here, we have developed a peptide antagonist of human angiogenin, which is a potent and tumor-associated angiogenic factor. ANI-E peptide was derived from the phage clone, which binds to angiogenin via the disulfide-constrained octapeptide epitope that is displayed on its surface, and is displaced by actin. Disulfide-constrained ANI-E peptide inhibits the interaction of angiogenin with actin, which is regarded as the angiogenin-binding protein on the surface of endothelial cells, without any visible effect on the ribonucleolytic activity of angiogenin. The peptide also inhibits the neovascularization that is induced by angiogenin in the chick chorioallantoic membrane assay. The antiangiogenic activity of the peptide is specific for angiogenin because the peptide does not have any apparent effect on embryonic angiogenesis or the preexisting blood vessels. The disulfide bond and the glutamic acid inside the disulfide ring of ANI-E peptide are indispensable for its antiangiogenin activity. Furthermore, ANI-E peptide blocks the angiogenesis that is induced by the angiogenin-secreting PC3 human prostate adenocarcinoma cells, without any direct effect on the proliferation, as well as the adhesion of PC3 cells to angiogenin. Therefore, the inhibition of the tumor-induced angiogenesis by ANI-E peptide is most likely caused by the neutralization of the extracellular angiogenin that is secreted by PC3 cells. On the basis of our results, ANI-E peptide may be effective for the treatment of various human tumors that secrete angiogenin. Our results also strongly support the hypothesis that the interaction of angiogenin with the cell surface actin-like protein is essential for the biological action of angiogenin, and angiogenin has an essential role in tumor-induced angiogenesis.