Skeletal muscle is a major component of body mass and plays a central role in the control of whole-body metabolism in humans and animals. Therefore, elucidation of the underlying mechanisms of skeletal growth and development are expected to lead to the discovery of novel genes and pathways related to muscle disease. miR-206, a skeletal muscle-specific microRNA, plays a crucial role in myogenesis; however, miR-206 is known to function in myogenic differentiation, whether or not it affects muscle cells' proliferation, and the underlying mechanisms are unknown. In this study, we investigated the effect of miR-206 on muscle cell proliferation and differentiation, as well as its effect on myofiber type conversion using mouse C2C12 myoblasts. The results showed that overexpression of miR-206 inhibited cell proliferation and promoted muscle cell differentiation, but it did not affect myofiber type conversion. Intriguingly, we found that overexpression of miR-206 suppressed muscle cell proliferation and induced cell cycle arrest in G0/G1 phase by inhibiting the glucose-6-phosphate dehydrogenase (G6PD) gene. Taken together, we demonstrated that the miR-206-G6PD pathway suppresses muscle cell proliferation, and these findings may facilitate the treatment of muscle diseases.-Jiang, A., Dong, C., Li, B., Zhang, Z., Chen, Y., Ning, C., Wu, W., Liu, H. MicroRNA-206 regulates cell proliferation by targeting G6PD in skeletal muscle.