Abstract: Objective: To study the anti-fatigue effect of irradiated Inonotus obliquus polysaccharide on mice. Method: ⁶⁰Co-γ rays were irradiated to obtain 5, 10, 20, 30 kGy irradiation doses and unirradiated Inonotus obliquus bacteria powder to produce polysaccharides. Divided into a control group, an irradiated high-medium-low-dose group, and a non-irradiated high-dose group. Male Kunming mice were given four weeks continuous gavage to determine the rod climbing, exhausted swimming time, changes in liver glycogen(LG), muscle glycogen(MG), blood lactate(BLA), lactate dehydrogenase(LDH), serum urea nitrogen(SUN), superoxide dismutase(SOD) and malondialdehyde(MDA) in skeletal muscle. Result: The optimal radiation dose measured in the experiment was 20 kGy. Each dose group had no significant effect on the body weight of the mice(P>0.05). The high and medium dose groups and the non-irradiated groups significantly prolonged the rod climbing time and exhausted swimming time of the mice(P<0.05). Irradiation of Inonotus obliquus polysaccharides increased the contents of liver glycogen and muscle glycogen in mice to the highest levels of 28.90 mg/g and 10.58 mg/g, reduced the contents of BLA, SUN and MDA in mice, and increased LDH and SOD activities in mice. The highest activity was 5308.80 U/L and 11.44 nU/mg. Moreover, LG, MG, LDH, SUN, SOD, MDA in the high-dose irradiated group were significantly different from those in the non-irradiated group(P<0.05), and there was no significant difference in BLA between the two groups(P>0.05). Conclusion: This test proves that the anti-fatigue effect of the irradiated Inonotus obliquus polysaccharide is further improved compared with the non-irradiated group, and that the irradiated Inonotus obliquus polysaccharide can relieve fatigue symptoms to a certain extent.