According to the known effects of each ingredient, Gan-Lu-Yin (GLY), a traditional Chinese herbal formula, has the potential to be an antiangiogenic agent. The purpose of this study was to explore the putative effect of GLY on antiangiogenesis. An ethanol extract of GLY was tested on chicken chorioallantoic membrane (CAM) and human umbilical vein endothelial cells (HUVEC) to evaluate the effects of GLY extract on cell proliferation, migration, and tube formation. The results showed that treatment with 1.0 mg/mL of GLY extract could markedly reduce cell migration and in vitro tube formation of HUVEC, and 1.5 mg/mL of GLY extract was sufficient to inhibit proliferation of HUVEC. The expression level of vascular endothelial growth factor (VEGF) of HUVEC was significantly decreased by 1.5 and 2.0 mg/mL of GLY extract. In chicken CAM assay, all tested concentrations of GLY extract were found to reduce the capillary mesh on the CAM of fertilized eggs. The inhibitory effects of GLY extract (1 mg/mL) were also found on tumor cell-induced HUVEC proliferation and tube formation. These observations suggested that GLY extract has an inhibitory effect on angiogenesis, which in turn may prevent tumor growth, and its mechanism might be partially associated with blocking VEGF protein expression of HUVEC.