Muscle fiber is the basic unit of skeletal muscle with strong self-adaptability, and its type is closely related to meat quality. Myod family inhibitor (Mdfi) has the function of regulating myogenic regulatory factors during cell differentiation, but how Mdfi regulates muscle fiber type transformation in myoblasts is still unclear. In the present study, we constructed overexpressing and interfering with Mdfi C2C12 cell models by lipofection. The immunofluorescence, quantitative real-time PCR (qPCR), and western blot results show that the elevated MDFI promoted mitochondrial biogenesis, aerobic metabolism and the calcium level by activating CaMKK2 and AMPK phosphorylation and then stimulated the conversion of C2C12 cells from fast glycolytic to slow oxidative type. In addition, after inhibiting IP3R and RYR channels, the higher MDFI reversed the blockage of calcium release from the endoplasmic reticulum by calcium channel receptor inhibitors and increased intracellular calcium levels. Therefore, we propose that the higher MDFI promotes muscle fiber types conversion through the calcium signaling pathway. These findings further broaden our understanding of the regulatory mechanism of MDFI in muscle fiber type transformation. Furthermore, our results suggest potential therapeutic targets for skeletal muscle and metabolic-related diseases.