Irpex lacteus is a white rot basidiomycete proposed for a wide spectrum of biotechnological applications. However, few studies examined its effects on exercise performance and physical fatigue. The present study evaluated the potential beneficial effects of I. lacteus extract (ILE) on physical fatigue in mice. Anti-fatigue activities of ILE were evaluated in Kunming mice using the forced swim test, rotating rod and forced running test. Serum and liver biochemical parameters were determined by an autoanalyzer and commercially available kits. Seven-day ILE administration at doses of 0.04, 0.2 and 1.0g/kg failed to influence mouse horizontal and vertical movement indicating its safety on the central nervous system. Compared with normal mice, ILE significantly increased persistent period during rotating rod and swimming tests, and reduced shock times in forced running test. Additionally, ILE resulted in 23.4% and 64.5% increments on adrenocorticotropic hormone and cortisol levels in serum. Compared with normal mice, and 209.0% increment on adenosine triphosphate level in liver (up to 2.5 mmol/gHb) were noted in ILE-treated mice. Moreover, ILE increased the level of super oxide dismutase and reduced the level of malondialdehyde in the liver suggesting its antioxidant activity. Data obtained from western bolt suggests that ILE-improved endurance capacity is mainly acquired through activating 5'-AMP-activated protein kinase (AMPK). ILE enhanced the endurance capacity of mouse by an elevation of antioxidant at least partially associated with AMPK pathway. Our data highlight the potential of I. lacteus as an antioxidant in the treatment of fatigue-related diseases.