Herba Epimedii, an herb commonly used in East Asian medicine, is commonly used for treatment of impotence, osteoporosis and many inflammatory conditions in traditional Chinese medicine. Recent studies revealed that Herba Epimedii also has anti-tumor or anti-cancer activities, which may possibly be mediated through anti-angiogenesis. This study aims to examine and confirm the anti-angiogenic activity in the herb using both in vivo and in vitro approaches. The 95% ethanol extract and four subsequent fractions (n-hexane, ethyl acetate (EA), n-butanol and aqueous fractions) of Herba Epimedii were tested on the zebrafish model by the quantitative assay for endogenous alkaline phosphatase; then, the active fraction was further tested on Tg(fli1a:EGFP)y1 zebrafish embryos and human umbilical vein endothelial cells (HUVECs) for the anti-angiogenic effects. In addition, the action mechanism of Herba Epimedii was further investigated on wild-type zebrafish embryos and HUVECs. The EA fraction showed anti-angiogenic effects in both in vivo and in vitro models. Further experiments demonstrated that it might affect angiogenesis by acting on multiple molecular targets in zebrafish embryos and ERK signaling pathway in HUVECs. In conclusion, Herba Epimedii can inhibit angiogenesis, which may be the mechanism for its anti-inflammatory, anti-tumor and anti-cancer actions. Copyright © 2012 John Wiley & Sons, Ltd.