Cucurbitacin-I is a naturally occurring tetracyclic triterpenoid compound with diverse physiological actions that include inducing apoptosis and blocking cell cycle progression of various cancer cells. However, its antiangiogenic activity has not yet been examined. Accordingly, we investigated the antiangiogenic efficacy and associated mechanisms of cucurbitacin-I in vitro using human umbilical vein endothelial cells (HUVECs). Cucurbitacin-I inhibited HUVEC proliferation, invasion, migration and tubule formation, as well as angiogenic activity by rat aorta explants. Notably, cucurbitacin-I inhibited phosphorylation of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1, which are key regulators of endothelial cell function and angiogenesis. In vivo matrigel plug assay in mice showed significant decrease in vascularization and hemoglobin content in the plugs from cucurbitacin-I-treated mice, compared with control mice. Overall, these results suggest that cucurbitacin-I inhibits various attributes of angiogenesis, which might contribute to its reported antitumor effects. Cucurbitacin-I warrants further investigation as an angiogenesis inhibitor for use in cancer treatment.