Oxidative stress–induced vascular endothelial cell (VEC) injury is a major mechanism in the initiation and development of atherosclerosis. Lunasin, a soybean-derived 43-aa peptide, has been previously shown to possess potent antioxidant and anti-inflammatory activities other than its established anticancer activities. This study investigated the effects of lunasin on protecting VECs from oxidative damage and inhibiting atherosclerotic plaque progression in apolipoprotein E–deficient (ApoE^?/?) mice and explored its underlying mechanism. Biochemical and histologic analyses were performed by using EA.hy926 human VECs and a high-fat diet (HFD) ApoE^?/? mouse atherosclerosis model. Our data indicated that lunasin attenuated H₂O₂-induced, mitochondria-dependent endothelial apoptosis via down-regulating Bax and up-regulating Bcl-2, inhibiting the mitochondrial depolarization, and reducing the release of cytochrome c, as well as decreasing the activation of casρa(bǔ)se-9 and caspase-3 in vitro and in vivo. Mechanic studies showed that lunasin significantly up-regulated heme oxygenase-1 via the PI3K/Akt/nuclear factor erythroid 2–related factor 2/antioxidant response element pathway, and reduced H₂O₂-induced ROS production in VECs, thereby attenuating oxidant-induced endothelial injury and inhibiting atherosclerotic plaque progression in ApoE^?/? mice. In conclusion, our in vitro and in vivo data suggest that lunasin protects VECs from oxidative damage by enhancing heme oxygenase-1 expression via activation of the PI3K/Akt/ nuclear factor erythroid 2–related factor 2/antioxidant response element pathway and inhibiting mitochondria-dependent apoptosis, thereby effectively attenuating atherosclerosis in HFD-fed ApoE^?/? mice. Lunasin may act as a potential therapeutic agent for the prevention and treatment of atherosclerosis.—Gu, L., Ye, P., Li, H., Wang, Y., Xu, Y., Tian, Q., Lei, G., Zhao, C., Gao, Z., Zhao, W., Tan, S. Lunasin attenuates oxidant-induced endothelial injury and inhibits atherosclerotic plaque progression in ApoE^?/? mice by up-regulating heme oxygenase-1 via PI3K/Akt/ Nrf2/ARE pathway. FASEB J. 33, 4836–4850 (2019). www.fasebj.org