Objective The aim of this study was to investigate the effects of hesperidin (HDN) on antioxidant activity in mice. Methods HDN scavenging free radicals was detected by spectrophotometry, inhibition of mitochondrial swelling was detected by pyrogallol autoxidation, and erythrocyte hemolysis was detected by Fe²⁺ phenanthroline. The mice were fed with HDN at different concentrations (0, 80, 160, 320 mg/kg) by gastric gavage for 12 days. ELISA and spectrophotometric methods were used to assay the amount of MDA in mouse liver and kidney tissues and the activity of antioxidant enzymes (SOD, CAT, GSH-PX), and the antioxidant enzyme gene mRNA expression was analyzed by RT-PCR. Results Compared with the control group, the radical (·OH, O₂^-·, DPPH·) clearance rate was significantly increased in the HDN groups. There was a significant decrease of oxidative hemolysis of erythrocytes and mitochondrial swelling in vitro. MDA content in the mouse liver and kidney tissues and serum showed a decrease, and the activity of antioxidant enzymes (SOD, CAT, GSH-PX) in the HDN group was significantly higher than that in the control group. There was an up-regulation of mRNA expression of antioxidant enzyme in mouse liver and kidney tissues. Conclusions The results showed that HDN can eliminate free radicals, reduce cell oxidative damage caused by free radicals, inhibit superoxide production, up-regulate antioxidant enzyme gene expression and enhance their enzyme activity, thus showing a good antioxidant effect.