During myogenesis, myoblast fusion depends on mitochondria and ion-channel-regulated intracellular Ca2+ signaling. However, the relationship between mitochondrial function and ion channels that affect calcium signaling in myoblasts is unknown. Here, we compared ionic currents associated with intracellular Ca2+ influx in normal and mitochondria-depleted (ρ0) L6 GLUT4myc myoblasts. In ρ0 myoblasts, we observed a decrease in the inward-rectifying K+ current (KIR2.1), which hyperpolarizes the membrane to generate the electromotive force for Ca2+ influx. The downregulation of KIR2.1 was induced by not only a decrease in KIR2.1 expression but also mitochondrial dysfunction of electron transport-coupled ATP synthesis in ρ0 myoblasts that might be affect intracellular Ca2+ signaling in ρ0 myoblasts. Our work elucidates the functional effect of mitochondria on myogenesis-related ion channels during myoblast differentiation.