Erxian Decoction (EXD), a traditional Chinese herbal formula, has been used to treat menopausal symptoms and other aging diseases for several decades. Recently, our laboratory found that EXD could inhibit the proliferation of breast cancer cells. This activity may be mediated by anti-angiogenic action. To investigate the anti-angiogenic activity of EXD, its inhibitory effect on blood vessel formation was evaluated using both wild type and transgenic zebrafish embryos with fluorescent vasculature in vivo. Both semi-quantitative and real-time quantitative polymerase chain reaction (qPCR) were carried out to evaluate the effect of EXD on the expression of several genes closely associated with angiogenesis in zebrafish. EXD was found to inhibit vessel formation in zebrafish embryos in a dose- and time-dependent manner. Furthermore, it reduced the mRNA expression of vascular endothelial growth factor A (VEGF-A) and the protein level of hypoxia inducible factor 1α (HIF-1α) in the embryos, suggesting the involvement of HIF-1 mediated VEGF-A signaling pathway in the anti-angiogenic action of EXD. The anti-angiogenic activity of EXD provides new insights to its clinical application and may in the future lead to the development of potential drugs for treating various cancers, especially in menopausal period.