Objective To elucidate the role of the glutathione (GSH) / glutathione peroxidase 4 (GPx4)-mediated ferroptosis pathway in preventing age-related hepatocyte peroxidation injury following aerobic exercise in mice, and to provide a new target for improving liver aging and metabolism disorders. Methods Twenty specific-pathogen-free C57BL/6 male mice aged 52 weeks were divided randomly into an elderly control group (EC group) and elderly exercise group (EE group) (n= 10 per group). The mice performed moderate-intensity exercise with incremental loads (1 ~ 2 weeks 14 m/ min, 3 ~ 4 weeks 15 m/ min, 5 ~ 10 weeks 16 m/ min, 11 ~ 16 weeks 17 m/ min, 60 min/ day, slope 0°) for 16 weeks. After perfusion of the ascending aorta, the lateral liver lobes were harvested and sectioned for hematoxylin and eosin staining and ultrathin sections were used for transmission electron microscopy. Levels of 8-hydroxy-2 deoxyguanosine (8-OHdG) and 4-hydroxynonenal (4-HNE) in the liver and serum interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay. Hepatic glycogen, triglycerides (TG), malondialdehyde (MDA), nicotinamide adenine dinucleotide phosphate (NADPH), and GSH were determined by colorimetry. Hepatic GPx4, glucose transporter (GLUT2), NAD(P)H: quinone oxidoreductase 1 (NQO1), and solute carrier protein 7 family member 11 (SLC7A11) were detected by Western Blot. Results (1)Oxidative damage to hepatocytes was effectively delayed, normal mitochondrial structure and glycogen storage in hepatocytes were maintained. (2) Hepatic GSH and NADPH contents were significantly increased in EE mice compared with EC mice (P< 0. 01). (3)In addition, liver levels of 8-OHdG, 4-HNE, MDA, and non-heme iron were significantly decreased in the EE group compared with the EC group (P< 0. 01). (4)Expression levels of GPx4, NQO1, and SLC7A11 in the liver were increased (P< 0. 01) while NOX2 expression was decreased (P< 0. 01) in the EE group compared with the EC group. Conclusions GSH synthesis was increased in aged mice following aerobic exercise, providing reaction substrates for GPx4 and activating the GSH/ GPx4 pathway. Ferroptosis was inhibited, thus improving hepatocyte peroxidation damage caused by aging, and maintaining the normal structure and physiological function of hepatocytes.