Rectal cancer is the second leading cause of cancer mortality in the western countries and accounts for 10% incidence and mortality of cancer in the whole world. Drug resistance and severe toxicity severely limited the efficiency of chemotherapy of rectal cancer. Oleanolic acid (OA) is a natural triterpenoid and an aglycone of many saponins. In the present study, we aimed to investigate the effect of OA on rectal cancer cell proliferation and its possible mechanism. We showed that OA concentration-dependently inhibited cell proliferation in HCT-15, HT-29, HCT-8 and Colo 205 human rectal cancer cell lines. OA significantly increased reactive oxygen species (ROS) generation and NADPH oxidase 2 (NOX2) expression in a concentration-dependent manner. In HCT-15 and HT-29 cells, siNOX2 notably suppressed OA-induced ROS generation, inhibition of cell proliferation, increase of S phase cell population and decrease of cyclin D1 and CDK2 expression. OA markedly decreased hypoxia-inducible factor 1α (HIF-1α) expression in HCT-15 and HT-29 cells in a concentration-dependent manner. Overexpression of HIF-1α significantly suppressed OA-induced inhibition of cell proliferation, increase of S phase cell population and decrease of cyclin D1 and CDK2 expression. Inhibition of NOX2 by siRNA notably blocked OA-induced suppression of HIF-1α expression. Our findings provide novel insights into OA-induced inhibition of rectal cancer cell proliferation and highlight NOX2/ROS/HIF-1α axis as a novel therapeutic target for the treatment of rectal cancer.