Calcium takes part in numerous cellular processes such as proliferation, migration, differentiation, or cell death and plays a particular role in myogenesis of skeletal muscle. Indeed, intracellular calcium signaling participates, in a non-negligeable manner, to the “ on ” signal of muscle differentiation from undifferentiated cells to differentiated myotubes. Therefore, this differentiation can be modulated by controlling calcium activity with electrical or optogenetic stimulation approaches. In this study, we used the optogenetic tool channelrhodopsin 2 (ChR2) to control calcium activity and to modulate skeletal muscle differentiation. Using primary cultures of mouse myotubes, we showed that ChR2 stimulation was well-adapted to control intracellular calcium activity at the single cell or whole culture scale. To modulate the calcium-dependent myotube differentiation, we used an optical stimulation protocol based on GCAMP6s-decoded spontaneous calcium activity patterns of differentiated myotubes. The optical training of myotubes increased the fusion index and their contractile ability. This study demonstrates that handling a mature calcium signature with such optogenetic tool improves the differentiation of primary murine myotubes.