Inonotus obliquus (IO) is used as functional food to treat diabetes. This study investigated the effect of IO supplementation on body composition in relation to changes in energy expenditure and exercise performance. Male Institute of Cancer Research mice were divided into four groups (n = 8 per group) and orally administered IO once daily for 6 wk at 0 (vehicle), 824 (IO-1×), 1648 (IO-2×), and 2472 mg/kg (IO-3×). IO supplementation increased muscle volume, exhaustive treadmill time, and glycogen storage in mice. Serum free fatty acid levels after acute exercise improved in the IO supplementation group, which exhibited changes in energy expenditure through the peroxisome proliferator-activated receptor (PPAR) pathway. RNA sequencing revealed significantly increased PPAR signaling; phenylalanine, ascorbate, aldarate, and cholesterol metabolism; chemical carcinogenesis; and ergosterol biosynthesis in the IO group compared with the vehicle group. Thus, IO supplements as nutraceuticals have a positive effect on lipid transport and exercise performance. In addition, this study was only IO supplementation without training-related procedures.