Introduction: The aim of this study was to evaluate gender differences in the oxidant/antioxidant response in different tissues of trained mice during physical stress with the hyperbaric oxygen preconditioning (HBOP) beforehand. Methods: Trained mice of both genders treated by HBOP were divided into three groups including basal, fasting and prolonged exercise. Parameters of oxidant/antioxidant state including nitric oxide (NO) were measured in blood and tissues. Gender differences in the effects of HBOP were analyzed in the basal levels, fasting and in the net response to exercise. Results: HBOP diminished the elevated basal levels of lipoperoxidation, NO, antioxidant enzymes and glutathione in liver of females only, compared to untreated group. A similar decrease in the basal levels of NO and enzymes was observed in other tissues of females as well. A strong decrease of basal level of glutathione in liver coincided to its increase in muscle, brain, small intestine and adipose tissue. Therefore, females after HBOP started prolonged exercise with a lower basal level of oxidative stress and antioxidant defense in liver but increased basal levels of glutathione in most tissues. Consequently, during exercise, females showed a strong net response in the liver and stability in muscle tissue, while in males the contrary response was found. Conclusion: Only in the tissues of females did HBOP drastically affect the basal levels of the oxidant/antioxidant status. Although performance decreased in both genders, it was better in females, likely related to the redistribution of glutathione from the liver to other tissues after HBOP.