Endothelial cell injury caused by reactive oxygen species (ROS) plays a critical role in the pathogenesis of atherosclerosis. Therefore, phytochemicals or antioxidants that inhibit the production of ROS have clinical value for the treatment of atherosclerosis. Rhein is one of the most important active components of rhubarb (Rheum officinale), a famous traditional Chinese remedy that possesses potent antioxidant properties through undefined mechanism(s). The aim of the present study was to determine whether rhein inhibits hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells (HUVECs). The oxidative injury model was established with H2O2. HUVECs were treated with different concentrations of rhein in the presence/absence of H2O2. The protective effects of rhein against the injury caused by H2O2 were evaluated. HUVECs incubated with 200 µmol/l H2O2 had significantly decreased cell viability, which was accompanied by cell apoptosis and upregulated Bid and caspase-3, -8 and -9 mRNA expression. Meanwhile, H2O2 treatment induced a marked increase in malondialdehyde (MDA) and lactate dehydrogenase (LDH) content and decreased the nitric oxide (NO) content and nitrogen oxide synthase (NOS), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity. However, pre-treatment with different rhein concentrations (2, 4, 8 and 16 µmol/l) significantly increased the viability of H2O2-injured HUVECs, decreased the MDA and LDH content, increased the NO content and NOS, SOD and GSH-PX activity in a dose-dependent manner and resulted in significant recovery from H2O2-induced cell apoptosis. In addition, the results of the qRT-PCR indicated that pre‑treatment with rhein downregulates the expression of Bid and caspase-3, -8 and -9 mRNA, which plays a key role in H2O2-induced cell apoptosis. The present study shows that rhein protects endothelial cells against oxidative injury induced by H2O2, suggesting that rhein is a potential compound for the prevention and treatment of atherosclerosis.