Background: Zhengyuan prescription (ZYP) is a Chinese herbal medicine used in clinical practice to protect against radiotherapy-induced injuries. In this study, we investigate the protective effect of ZYP against X-ray-induced injury of human umbilical vein endothelial cells (HUVECs), and we explore the mechanisms underlying this effect.Methods: After 3 h of ZYP intervention, the cells in the ZYP group were irradiated with 6 Gy X-rays and cultured for 48 h. Subsequently, the cell viability, cell morphology, mitochondrial membrane potential, and apoptosis and oxidative stress markers were observed, as well as the expressions of apoptotic and oxidative stress proteins.Results: The obtained results demonstrate that exposure to X-rays promotes cell death, reduces mitochondrial membrane potential, and induces the pirroduction of intracellular reactive oxygen species (ROS). Pretreatment with ZYP reverses these effects to a great extent. Moreover, it up-regulates the expression of the B-cell lymphoma 2 (Bcl-2) apoptosis inhibitor protein while down-regulating the expressions of Bcl-2-associated X protein (Bax), caspase-3, and caspase-9. Interestingly, ZYP can also inhibit oxidative stress injury by activating the expression of Nrf2 (Nuclear Factor E2 related factor) regulated antioxidant enzyme genes such as Heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1)Conclusions: This study is the first to demonstrate that ZYP suppresses X-ray-induced injury of HUVECs by activating the Nrf2 signaling pathway.