Homothermal animals need to keep their body temperature within a narrow range. Only a few degrees Celsius change in temperature has a dynamic influence on many physiological processes. To investigate the effect of the body temperature on muscle cell differentiation, we cultured the mouse myoblast cell lines C2C12 and Sol8 at lower temperatures than mouse body temperature. At 38°C, the cells fused into multinucleated myotubes within 4 days after the induction of differentiation. However, myotube formation was blocked at 30°C, whereas it was delayed but relatively normal at 35°C. The myoblasts expressed MyoD, but not myogenin, at 30°C. Id3, which acts as a negative regulator of myogenic regulatory factors (MRFs), was expressed at a higher level at 30°C than at 38°C, whereas the expression level of E2A, which acts as a positive regulator of MRF expression, exhibited no difference between these temperatures. We also found that the expression of muscle-enriched microRNAs decreased at 30°C. In addition, we investigated the expressions of MyoD and myogenin during mouse satellite-cell activation in single-fiber culture as an in-vivo model, and found that the expression of myogenin, but not of MyoD, was inhibited. These results suggest that skeletal muscle formation can be regulated by temperature, and that the physiological body temperature plays a crucial role in the myogenesis of homothermal animals.