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Objective: To explore the effects of different concentrations of Rosa roxburghii Tratt powder on antioxidant stress injury of skeletal muscle in overtrained rats. Methods: The rats were divided into the control group (C group), exercise group (HT group), low concentration Rosa roxburghii Tratt powder exercise group (L-HT group) and high concentration Rosa roxburghii Tratt powder exercise group (H-HT group). HT, L-HT and H-HT groups underwent treadmill training with increasing load for 6 weeks, respectively. During the training, L-HT group and H-HT group were intragastrically administered with 20 and 50 mg/mL Rosa roxburghii Tratt powder solution at the dose of 20 mL·kg−1·d−1, respectively. Group C and HT were given the same dose of saline. The samples collected 24 hours after last training were used to measure serum creatine kinase (CK), interleukin -1β (IL-1β) and tumor necrosis factor-α (TNF-α). The activities of superoxide dismutase (SOD) and the contents of malondialdehyde (MDA) and glutathione (GSH) in skeletal muscle were determined. The expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1(HO-1), B cell lymphoma factor-2 (Bcl-2) and Bcl-2-related X protein(Bax) in skeletal muscle was detected. Results: Compared with the HT group, the activity of CK decreased (P<0.05), the activity of SOD increased (P<0.05), the contents of IL-1β, TNF-α and MDA decreased (P<0.05), the concentration of GSH increased (P<0.05), the expressions of Nrf2, HO-1 and Bcl-2 proteins increased (P<0.05), and the expression of Bax protein decreased in L-HT group (P<0.05). The activity of CK decreased (P<0.01), the activity of SOD increased (P<0.01), the contents of IL-1β, TNF-α and MDA decreased (P<0.01), the concentration of GSH increased (P<0.01), the expressions of Nrf2, HO-1 and Bcl-2 proteins increased (P<0.01), and the expression of Bax protein decreased in H-HT group (P<0.01). Compared with the L-HT group, the activity of CK decreased (P<0.05), the activity of SOD increased (P<0.05), the contents of IL-1β, TNF-α and MDA decreased (P<0.05), the expression of Nrf-2, HO-1 and Bcl-2 protein increased (P<0.05), and the expression of Bax protein decreased in H-HT group (P<0.05). Conclusion: Rosa roxburghii Tratt powder had a good protective effect on oxidative stress injury induced by overtraining in rats, and there was a dose-response relationship between the dosage of Rosa roxburghii Tratt powder and antioxidative stress injury in a certain range.