Endothelial cells (ECs), that comprise the tumor vasculature, are critical targets for anticancer radiotherapy. The aim of this work was to study the mechanism by which SU5416, a known anti-angiogenesis inhibitor, modifies the radiation responses of human vascular ECs. Two human endothelial cell lines (HUVEC and 2H11) were treated with SU5416 alone, radiation alone, or a combination of both. In vitro tests were performed using colony forming assays, FACS analysis, western blotting, immunohistochemistry, migration assay, invasion assays and endothelial tube formation assays. The combination of radiation and SU5416 significantly inhibited cell survival, the repair of radiation-induced DNA damage, and induced apoptosis. It also caused cell cycle arrest, inhibited cell migration and invasion, and suppressed angiogenesis. In this study, our results first provide a scientific rationale to combine SU5416 with radiotherapy to target ECs and suggest its clinical application in combination cancer treatment with radiotherapy.