MicroRNAs (miRNAs) are endogenous short non-coding RNAs and exert their function by targeting mRNAs of genes. miRNA-206 (miR-206) is exclusively expressed in adult skeletal muscles and plays an important role in myogenesis. However, the regulatory mechanisms of miR-206 in myoblasts proliferation and differentiation are still limited. In this study, we validated that Notch3 is a direct target gene of mouse miR-206 using dual-luciferase reporter assay and miR-206-overexpressed experiments. Furthermore, we demonstrated that the negative effect of overexpression of miR-206 and knockdown of Notch3 on C2C12 cells proliferation, and found that the effect was produced by controlling the transition of G0/G1 and S phase in C2C12 cell cycle. In summary, our results provide direct evidences that miR-206 regulates skeletal muscle cells proliferation and cell cycle arrest partly by targeting the Notch3 gene, which strengthens our understanding of regulatory mechanisms on miR-206 in skeletal muscle growth and development.