MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules about 22 nucleotides in length and are encoded by endogenous genes, and are involved in the regulation of post-transcriptional gene expression in animals and plants. Many studies have shown that microRNAs regulate the development of skeletal muscle, mainly manifested in the activation of muscle satellite cells and biological processes such as proliferation, differentiation, and formation of muscle tubes. In this study, miRNA sequencing screening of longissimus dorsi (LD, mainly fast-twitch fibers) and soleus muscle (Sol, dominated by slow-twitch fibers) identified the miR-196b-5p as a differentially expressed and highly conserved sequence in different skeletal muscles. Studies of miR-196b-5p in skeletal muscle have not been reported. In this study, miR-196b-5p mimics and inhibitor were used in miR-196b-5p overexpression and interference experiments in C2C12 cells. The effect of miR-196b-5p on myoblast proliferation and differentiation was analyzed by western blotting, real-time quantitative RT-PCR, flow cytometry, immunofluorescence staining, and the target gene of miR-196b-5p was identified by bioinformatics prediction and analyzed by dual luciferase reporter assays. The results showed that overexpression of miR-196b-5p could significantly increase the mRNA and protein expression of Cyclin B, Cyclin D and Cyclin E (P<0.05); Cell cycle analysis showed that overexpression of miR-196b-5p significantly increased the proportion of cells in the S phase (P<0.05), indicating that miR-196b-5p could accelerate cell cycle progress. Results of EdU staining showed that overexpression of miR-196b-5p significantly promoted cell proliferation. Conversely, inhibition of miR-196b-5p expression could significantly reduce the proliferation capacity of myoblasts. Further, overexpression of miR-196b-5p could significantly increase the expression levels of myogenic marker genes MyoD, MoyG and MyHC (P<0.05), thereby promoting myoblast fusion and accelerating C2C12 cell differentiation. Bioinformatics predictions and dual luciferase experiments demonstrated that miR-196b-5p could target and inhibit the expression of the Sirt1 gene. Altering the Sirt1 expression could not rescue the effects of miR-196b-5p on the cell cycle, but could weaken the promoting effects of miR-196b-5p on myoblast differentiation, suggesting that miR-196b-5p promoted myoblast differentiation by targeting Sirt1.