Objective To investigate the effect of hyperbaric oxygen preconditioning on exercise-induced fatigue in rats. Methods SD rats were subjected to loaded swimming after hyperbaric oxygen (250 kPa, φ_B[O₂]≥99%) pretreatment, and rats exposed to normal pressured air were set as air control group. The time to exhaustion and levels of lactic acid (LA), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum and skeletal muscle were observed to assess the effect of hyperbaric oxygen preconditioning on exercise-induced fatigue in rats. Results The time to exhaustion in loaded swimming of rats in hyperbaric oxygen pretreatment group was significantly prolonged compared with that in the air control group ([56.13±16.91] min vs[23.13±9.85] min, P<0.01). After swimming for 20 min, the LA and MDA levels in the skeletal muscle and serum were significantly lower in the hyperbaric oxygen pretreatment group compared with air control group (P<0.01), while the SOD levels in skeletal muscle and serum were significantly higher compared with air control group (P<0.01). Conclusion Hyperbaric oxygen pretreatment can significantly delay the exercise-induced fatigue in rats.