Calcineurin B homologous protein3 (CHP3) is an EF-hand calcium-binding protein. In our previous study, we found that the expression level of endogenous CHP3 protein was increased in an early differentiation step in C2C12 mouse myoblasts. In this study, we aimed to elucidate the role of CHP3 in skeletal muscle differentiation. Live-cell time-lapse imaging revealed that CHP3 over-expression inhibited cell division of undifferentiated C2C12. Therefore, CHP3 may negatively regulate the proliferation of C2C12.