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Diabetes mellitus (DM) is a metabolic disease characterized by high blood sugar. Due to its complex pathogenesis, no effective drugs have been found so far. Ophiopogonin D (OP-D) has anti-inflammatory, antioxidant, and anticancer activities, but its role in DM has not been studied so far. Hydrogen peroxide (H2O2) was used to induce INS-1 cells. INS-1 cells induced by H2O2 were treated with OP-D, and cell apoptosis, oxidative stress damage, and related indexes of mitochondrial function were respectively detected by cell counting kit-8, flow cytometry, western blot, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, JC-1 fluorescent probe, and related kits. Subsequently, molecular docking techniques were used to investigate the relationship between OP-D and Keap1 and to explore the regulation mechanism of OP-D on H2O2-induced oxidative stress and mitochondrial function in INS-1 cells. OP-D inhibited the apoptosis and oxidative stress level of H2O2-induced INS-1 cells, thereby inhibiting cell damage. Moreover, OP-D inhibited mitochondrial dysfunction in H2O2-induced INS-1 cells. At last, we found that Keap1/Nrf2 specific signaling pathway inhibitor ML385 was able to reverse the inhibitory effect of OP-D on H2O2-induced oxidative stress and mitochondrial dysfunction in INS-1 cells. In conclusion, OP-D improves oxidative stress and mitochondrial dysfunction in pancreatic β cells induced by H2O2 through activating Keap1/Nrf2/ARE pathway in DM.