MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial regulatory roles in many biological processes, including the growth and development of skeletal muscle. miRNA-100-5p is often associated with tumor cell proliferation and migration. This study aimed to uncover the regulatory mechanism of miRNA-100-5p in myogenesis. In our study, we found that the miRNA-100-5p expression level was significantly higher in muscle tissue than in other tissues in pigs. Functionally, this study shows that miR-100-5p overexpression significantly promotes the proliferation and inhibits the differentiation of C2C12 myoblasts, whereas miR-100-5p inhibition results in the opposite effects. Bioinformatic analysis predicted thatTrib2has potential binding sites for miR-100-5p at the 3′UTR region. A dual-luciferase assay, qRT-qPCR, and Western blot confirmed thatTrib2is a target gene of miR-100-5p. We further explored the function ofTrib2in myogenesis and found thatTrib2knockdown markedly facilitated proliferation but suppressed the differentiation of C2C12 myoblasts, which is contrary to the effects of miR-100-5p. In addition, co-transfection experiments demonstrated thatTrib2knockdown could attenuate the effects of miR-100-5p inhibition on C2C12 myoblasts differentiation. In terms of the molecular mechanism, miR-100-5p suppressed C2C12 myoblasts differentiation by inactivating the mTOR/S6K signaling pathway. Taken together, our study results indicate that miR-100-5p regulates skeletal muscle myogenesis through theTrib2/mTOR/S6K signaling pathway.