Objective: To investigate the beneficial effects of astragaloside IV (AS-Ⅳ) on oxidative stress injury induced by hydrogen peroxide (H2O2) in cultured human aortic endothelial cells (HAECs). Methods: HAECs were cultured in vitro and randomly divided into Control group, H2O2 treated group，and AS-Ⅳ treated group. The cells were treated with H2O2（400 μmol/L) for 36 hours as H2O2 group. For AS-Ⅳ treatment, the cells were first treated with H2O2 for 12 hours, then AS-Ⅳ(50 μg/mL) were added into culture medium for 24 h. After 36 h, all the cells were examined for oxidative stress injury and mitochondrial antioxidative function. Results: Compared with the Control cells, the addition of H2O2 significantly increased the MDA content, NADPH oxidase activity, and mitochondrial ROS content of the cells, whereas the SOD activity, Mn-SOD activity and mitochondrial transmembrane potential level were significantly decreased. The administration of AS-Ⅳ significantly decreased NADPH oxidase activity, MDA content and mitochondrial ROS content, and increased SOD activity, Mn-SOD activity and mitochondrial transmembrane potential level, compared with those of H2O2 treated injured cells. Conclusion: In the present study, AS-Ⅳ attenuates H2O2 induced oxidative stress in HAECs. The enhancement of mitochondrial antioxidant function by AS-Ⅳ may be one of the possible pathways of AS-Ⅳ to protect HAECs from oxidative stress.