MicroRNAs (miRNAs) are a class of conserved non-coding RNAs that are widely regarded as important regulators in a variety of biological processes. Increasing evidence has revealed that skeletal muscle fiber-type conversion is regulated by miRNAs, but the molecular mechanism is still not fully understood. In this study, we confirmed the role of miR-22-3p on skeletal muscle fiber-type conversion and investigated its potential mechanism in C2C12 myotubes. Here, we found that the miR-22-3p mimics inhibited the expressions of myosin heavy chain I (MyHC I), MyHC IIa and promoted the expression of MyHC IIb, while miR-22-3p inhibitor got inverse results. miR-22-3p mimics also downregulated phosphorylated AMPK, SIRT1 and PGC-1ɑ protein levels, which control the expression of oxidative fiber-related genes. Furthermore, Compound C (AMPK inhibitor) eliminated the effect of miR-22-3p inhibitor on MyHC I, MyHC IIa and MyHC IIb expressions. However, AICAR (AMPK activator) also abolished the effect of miR-22-3p mimics on MyHC I, MyHC IIa and MyHC IIb expressions. Collectively, our results suggest that miR-22-3p regulates skeletal muscle fiber-type conversion through inhibiting AMPK/SIRT1/PGC-1ɑ signaling pathway.