Skeletal muscle, which is crucial for meat production, color, and quality is regulated by complex genetic mechanisms. MicroRNAs (miRNA), serve a crucial part in regulating skeletal muscle myogenesis together with the switching of muscle fiber types, but the identification of key miRNAs and their underlying molecular mechanism has been hindered. In the present study, miRNA sequencing was utilized to identify the differentially expressed miRNAs (DEMs) in different skeletal muscles, among which miR-196-5p was found notably upregulated in chicken soleus (SOL) muscles, suggesting the potential role of miR-196-5p in slow-twitch fiber formation. Next, the gain- and loss-of-function experiments confirmed the inhibitory role and stimulatory effects of miR-196-5p on myoblast expansion, myotube maturation, and slow-twitch myofibers formation, respectively. Through integrated bioinformatics and experimental analysis, the interaction between miR-196-5p and PBX1 was additionally clarified. PBX1 exhibits a promotive role in skeletal muscle myogenesis, while it exerts an inhibitory effect during the formation of slow-twitch myofibers. In conclusion, we propose that miR-196-5p has an important involvement in modulating of skeletal muscle structural composition and function.