Apoptosis of human vein endothelium cell caused by reactive oxygen species is implicated in the pathogenesis of cardiovascular diseases. Rutin, an active flavonoid compound, is well known to possess potent antioxidant properties against oxidative stress insults through undefined mechanism(s). In this study, we first investigated the possible protective effects of rutin against apoptosis of human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H(2)O(2)) and the associated signaling pathways. Decreased viability and increased apoptosis were observed in the HUVECs incubated with 200microM H(2)O(2) for 12h. By examining the effect of rutin on H(2)O(2)-induced apoptosis in HUVECs, we found that rutin pretreatment significantly attenuated H(2)O(2)-induced apoptosis in HUVECs. We next examined the signaling involved in rutin-mediated anti-apoptotic effects. It was found that rutin pretreatment attenuated excessive reactive oxygen species in HUVECs exposed to H(2)O(2). Rutin also prevented the increased DNA fragment formation and glutathione (GSH) depletion and inhibited the collapse of mitochondrial membrane potentials (DeltaPsim) that occurred in HUVECs exposed to H(2)O(2), which protected HUVECs against oxidative damage and the further mitochondrial membrane integrity impairment, leading to apoptosis. In conclusion, the results suggested that rutin (50microM) blocked apoptosis in HUVECs through decreasing reactive oxygen species, increasing GSH, restoring DeltaPsim and thus protecting DNA damage. Our research indicated that rutin protected the intracellular GSH antioxidant system and prevented H(2)O(2)-induced apoptosis of HUVECs through regulating reactive oxygen species mediated mitochondrial dysfunction pathway.