One of the most dynamic processes during skeletal myogenesis is cell-cell fusion, which promotes skeletal myoblast to form a large multi-nucleated myofiber equipped with EC-coupling machinery. Recently discovered transmembrane protein Myomixer (Mymx) executes cell-cell fusion, although the physiological significance of Mymx-mediated fusion on myogenic cell differentiation remains largely unknown. The current study focuses on the intracellular Ca²⁺ signaling mechanism to understand the physiological importance of Mymx mediated cell fusion in regulating voltage-induced Ca²⁺release in the developing skeletal muscle cells. C2C12 cells were used as an in vitro myogenesis model of skeletal muscle cells. We investigated intracellular Ca²⁺ release upon electric field stimulation applied to differentiated Mymx-KO cells and Mymx-rescued cells. We found that the efficiency of Ca²⁺ response was dependent on the Mymx gene expression. Importantly, expression levels of MyoD and myogenin were almost unaltered by the gene rescue of Mymx, suggesting that the Mymx-dependent Ca²⁺ response is regulated independently of these transcription factors. In conclusion, we found a novel regulatory linkage between Mymx expression and the voltage-induced Ca²⁺ release essential for EC coupling.