Objective To elucidate the role of Glutathione (GSH)/Glutathione peroxidase 4 (GPx4) -mediated ferroptosis pathway in preventing age-related hepatocyte peroxidation injury by aerobic exercise in mice, and to provide a new target for improving liver aging and metabolism disorders. Methods 20 SPF C57BL/6 male mice aged 52 weeks were randomly divided into the elderly control group, (EC) and the elderly exercise group (EE), with 10 mice in each group. The mice performed 16 w moderate intensity exercise with the incremental load (1~2 w 14 m/min, 3~4 w 15 m/min, 5~10 w 16 m/min, 11~16 w 17 m/min, 60 min/d, slope 0?. After perfusion of the ascending aorta, the lateral lobes of the liver were harvested for HE sections and ultrathin transmission electron microscope sections. The levels of 8-hydroxy-2 deoxyguanosine (8-OHdG), 4-Hydroxynonenal (4-HNE) in liver and serum interleukin-6 (IL-6) were detected by ELISA. Hepatic glycogen, triglyceride (TG), malondialdehyde (MDA), nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione (GSH) were determined by colorimetry. Hepatic GPx4, glucose transporter (GLUT2) and NAD(P)H:quinone oxidoreductase 1(NQO1) and solute carrier protein 7 family member 11 (SLC7A11) were detected by Western blot. Results ① The oxidative damage of hepatocytes in elderly exercise mice were effectively delayed, and the normal structure of mitochondria and glycogen storage in hepatocytes were maintained. ② Compared with the elderly control group, the content of hepatic GSH and NADPH in the elderly exercise group were increased significantly (P<0.01). ③ Compared with the elderly control group, 8-OHdG, 4-HNE, MDA and non-heme iron in liver of the elderly exercise group were decreased significantly (P<0.01). ④ The expression of GPx4, NQO1 and SLC7A11 in the liver of the elderly exercise group were increased (P<0.01), while the expression of NOX2 was decreased (P<0.01). Conclusions The synthesis of GSH was increased in aged mice after aerobic exercise, which provided sufficient reaction substrates for GPx4, and GSH /GPx4 pathway was activated. The ferroptosis process was inhibited, which improved hepatocyte peroxidation damage caused by aging, and maintained the normal structure and physiological function of hepatocytes.