Genistein (Gen), a soy isoflavone, is considered to exert potent antitumor effect partially through its anti-angiogenesis property. However, the precise molecular mechanism is still unknown. Our previous investigations have demonstrated that genistein down-regulates expression of pro-angiogenic factors via inhibiting protein tyrosine kinase (PTK) activity both in breast cancer cells and in xenograft tumors. In the present experiment, we chose cultured human umbilical vein endothelial cells (HUVECs), which have a considerable role in tumor angiogenesis formation, to explore the influence of genistein on VEGF-stimulated endothelial cell activation and the underlying mechanism. Stimulation of human primary HUVECs by VEGF not only increased endothelial cell protein tyrosine kinase (PTK) activity but also augmented matrix metalloproteinase-2 (MMP-2), -9 secretions and increased MMP-2, -9 activities. Treatment of ECs with genistein induced VEGF-loaded endothelial apoptosis by inhibiting production and activity of matrix metalloproteinases (MMPs). In addition, exposure to genistein decreased activation of JNK and p38, not ERK-1/2, induced by VEGF. Collectively, our findings suggested that the inhibition of PTK activity and MAPK activation and the decrease in MMPs production and activity by genistein interrupt VEGF-stimulated endothelial cell activation, which thereby may represent a mechanism that would explain the anti-angiogenesis effect of genistein and its cancer-protective function.