Grifola frondosa, a large edible mushroom also known as maitake, has been used as a health food for centuries in China and Japan. In the present study, we examined anti-angiogenic activity of a water extract of the fruiting body of G. frondosa (GFW). An in vivo angiogenesis assay using chick chorioallantoic membrane revealed that GFW (1-100 microg/mL) dose-dependently inhibited vascular endothelial growth factor (VEGF)-induced angiogenesis. In addition, GFW inhibited VEGF-induced proliferation, chemotactic migration, and capillary-like tube formation of human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. Upon stimulation by VEGF, HUVECs rapidly increased reactive oxygen species production, which was significantly blocked by the treatment with GFW. Moreover, phosphorylation of extracellular signal-regulated kinase 1/2, a downstream signaling molecule following VEGF receptor activation, was also inhibited by GFW. The results indicate that GFW effectively inhibit angiogenesis by blocking VEGF signaling and suggest that G. frondosa fruiting body may be a valuable medicinal food for treatment of angiogenesis-associated human diseases.