Abstract: Objective: To study the anti-fatigue effect of Gastrodia powder on mice. Methods: Mice were divided into a blank control group and groups receiving low (0.25 g/kg), medium (0.5 g/kg), and high (1 g/kg) doses of Gastrodia powder via continuous intragastric administration for 30 days. The body weight of mice was recorded during the period. One hour after the last intragastric administration, the swimming exhaustion time and organ index, and the blood urea nitrogen (BUN), blood lactic acid (BLA), muscle glycogen (MG), liver glycogen (LG) content, total superoxide dismutase (T-SOD) activity, malondialdehyde (MDA) content, lactate dehydrogenase (LDH), and creatine kinase (CK) activities were detected. Results: Compared with the blank group, the Gastrodia powder high-dose group extended the swimming time from 23.31 min to 46.97 min (P<0.01), reduced the BUN and BLA contents (P<0.01), and significantly increased the MG and LG contents (P<0.01) with 0.33 and 2.68 mg/g, respectively, improved the endurance of mice. In addition, Gastrodia powder had antioxidant effects. The high-dose group increased the activity of T-SOD (P<0.05) and reduced the levels of MDA (P<0.05), LDH (P<0.05) and CK (P<0.01) in the serum. Conclusion: The effect of Gastrodia powder on fatigue mice was dose-dependent, and 1g/kg could significantly improve the body's anti-fatigue ability.