The aim of this study was to evaluate the effects of ardipusilloside I isolated from Ardisia pusilla on tumor angiogenesis and its mechanism of action. The anti-angiogenic effect in vivo was evaluated on xenograft in the athymic mice model and the chicken chorioallantoic membrane (CAM) neovascularization model, the inhibition of growth in vitro was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and the mechanism was demonstrated through detecting microvessel density (MVD), vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) and P-VEGFR2 protein expressions, as well as mRNA expressions of VEGF and VEGFR2. The results showed that ardipusilloside I had a good inhibitory effect on A549 xenografted tumor growth, angiogenesis of CAM, and A549 cell growth. Compared to the negative control, MVD protein and mRNA expressions of VEGF and VEGFR were significantly inhibited by ardipusilloside I in a dose-dependent manner. These findings suggested that ardipusilloside I might be a promising candidate as angiogenesis inhibitors.