Calcineurin B homologous protein 3 (CHP3) is a calcium-binding protein and is highly expressed in heart. We previously found that this protein inhibits cardiomyocyte hypertrophy. However, the function in skeletal muscle is unknown. In the present study, we examined its role in the differentiation and fusion process of mouse skeletal muscle. CHP3 was barely expressed in adult rectus femoris muscle, but abundantly expressed in neonatal muscle. The expression level of CHP3 was increased in mouse C2C12 myoblasts during the differentiation into myocytes. Disruption of the CHP3 gene in the myoblasts with the CRISPR-Cas9 system decreased the expression level of myosin heavy chain (MHC), a marker of skeletal muscle differentiation. In addition, the myotube fusion index (number of myotubes with at least two nuclei per total myotubes) was decreased. On the other hand, over-expression of CHP3-mCherry increased MHC expression and myotube fusion. These results suggest that CHP3 regulates skeletal muscle development.