Skeletal muscle atrophy impairs quality of life. However, there is no effective medications. To maintain muscle mass, it is crucial to enhance differentiation of skeletal muscle stem cells and protein synthesis in myofibers. Our search using C2C12 cells determined that DCEBIO, a small/intermediate conductance Ca2+ activated K+ (SK/IK) channel opener promotes myogenic differentiation and muscle hypertrophy. DCEBIO hyperpolarized myoblast membrane potential and increased intracellular Ca2+ concentration. The expression of myogenin was elevated. DCEBIO treatment increased phosphorylation level of S6K, but not that of Akt. DCEBIO did not alter the expression of atrophy and hypertrophy related genes. SK/IK channel blocker, apamin (100 nM) and TRAM-34 (1 μM) inhibited myogenic differentiation but did not attenuate hypertrophic effect of DCEBIO in C2C12 cells. These results suggest that DCEBIO enhances myogenic differentiation by opening of SK/IK channel and activating myogenic regulatory factor. In contrast, DCEBIO increases muscle mass by activating S6K independent of SK/IK channel activation. This is a significant study for future drug development for muscle atrophy.