Although angiogenesis inhibitors targeting VEGF/VEGFR2 have been applied for tumor therapy, the outcomes are still unsatisfactory. Thus, it is urgent to develop novel angiogenesis inhibitor for cancer therapy from new perspectives. Identification of novel angiogenesis inhibitor from natural products is believed to be one of most promising strategy. In this study, we showed that pristimerin, an active agent isolated from traditional Chinese herbal medicine Celastrus aculeatus Merr, was a novel tumor angiogenesis inhibitor that targeting sonic hedgehog (Shh)/glioma associated oncogene 1 (Gli1) signaling pathway in non-small cell lung cancer (NSCLC). We showed that pristimerin affected both the early- and late-stage of angiogenesis, suggesting by that pristimerin inhibited Shh-induced endothelial cells proliferation, migration, invasion as well as pericytes recruitment to the endothelial tubes, which is critical for the new blood vessel maturation. It also suppressed tube formation, vessel sprouts formation and neovascularization in chicken embryo chorioallantoic membrane (CAM). Moreover, it significantly decreased microvessel density (MVD) and pericyte coverage in NCI-H1299 xenografts, resulting in tumor growth inhibition. Further research revealed that pristimerin suppressed tumor angiogenesis by inhibiting the nucleus distribution of Gli1, leading to inactivation of Shh/Gli1 and its downstream signaling pathway. Taken together, our study showed that pristimerin was a promising novel anti-angiogenic agent for the NSCLC therapy and targeting Shh/Gli1 signaling pathway was an effective approach to suppress tumor angiogenesis.