In this study, we determined the protective effect of total flavonoids from Spirodela polyrrhiza (L.) Schleid (STF), which is a kind of traditional Chinese medicine, on human umbilical vein endothelial cells (ECV-304) damage induced by hydrogen peroxide (H(2)O(2)). Treated with 1mmol/L H(2)O(2) for 1h, the viability of ECV-304 cells markedly decreased. However, pretreatment with 10-50mug/mL STF resulted in a significant recovery. The survival rate of ECV-304 increased from 21.98% (only treated with 1mmol/L H(2)O(2)) to 64.74% (pretreated with 50microg/mL STF), which accompanied with the amounts of malondialdenhyde (MDA) decreasing from 1.6883nmol/L to 0.9628nmol/L. Furthermore, compared with control group, the 50mumol/L STF pretreatment enhanced the total antioxidant capacity (T-AOC) by 4.49 times, increased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) by 85.12%, 158.94% and 94.5%,respectively, and increased the content of nitric oxide (NO) by 116.55%. Taken together, STF protect ECV-304 cells against H(2)O(2) damage by enhancing the antioxidant ability and increasing NO production.