Angiogenesis is central to the growth of cancers and VEGFR-1/Flt-1 plays an important role during the neovascularization under pathological conditions. We previously founded a VEGFR1 antagonistic peptide, F56, by screening the phage peptide library. We showed that DHFR-F56 chimeric protein displayed anti-tumor activity and inhibited angiogenesis, however the anti-tumor activity of monomeric F56 and the mechanism remain unclear. In this study, we found that the F56 didn't affect VEGF-A induced endothelial cell proliferation, but reduced migration and tube formation of endothelial cells. F56 also inhibited the sprout of rat aortic endothelial cells, the angiogenesis of chicken embryo chorioallantoic membrane as well as the generation of subintestinal vein vessels (SIV) in zebrafish embryos. We found that F56 inhibited VEGF-induced phosphorylation of VEGFR1, as well as the phosphorylation of the downstream of PI3K-AKT axis. However, F56 had no effect on the phosphorylation of VEGFR2. Correlating with these effects, F56 inhibited xenograft growth of HT-29 and MGC-823 cells in BALB/c nude mice, and significantly suppressed the lung metastasis of B16 cells in C57BL/6 mice. Our study demonstrated that monomeric peptide F56 had a significant anti-tumor activity by inhibiting angiogenesis, and laid the foundation for its clinical application.