The aim of the present study was to examine whether transient receptor potential melastatin 2 (TRPM2) plays a role in muscle fiber type transition during exercise. Mice were trained at a speed of 12 m/min at a slope of 0 ° for 60 min 5 consecutive d/wk for 4-wk. Exhaustion tests were performed on the treadmill (the speed was set at 6 m/min at a slope of 0° and increased at a rate of 1 m/min every 6 min). Isolated primary skeletal muscle cells from TRPM2 knockout (KO) mice showed lower amplitudes of electrical stimuli (ES)-induced Ca2+ signals when compared to wild-type (WT) mice, due to a defect in Ca2+ influx. Moreover, TRPM2 KO mice had a higher proportion of fast-twitch skeletal muscle fibers and a lower proportion of slow-twitch muscle fibers before exercise than WT mice. After exercise, the expression of slow-twitch skeletal muscle fibers was increased only in WT mice, but not in TRPM2 KO mice. ES-induced nuclear translocation of the Ca2+-dependent transcription factor NFATc1 was significantly lower in TRPM2 KO mice than WT mice. TRPM2 KO mice also showed decreased mitochondrial Ca2+ and membrane potential. Lactate levels were higher in the skeletal muscle cells of TRPM2 KO mice before and after ES, compared to WT mice. Collectively, these data indicate that TRPM2-mediated Ca2+ signaling plays a critical role in the regulation of fiber type switching and mitochondrial function in skeletal muscle.