Calcineurin B homologous protein3 (CHP3) is an EF-hand calcium-binding protein. In previous studies, we found that it is largely expressed in rat cardiomyocytes. Silencing of its expression by RNAi increased the cell size and induced the phosphorylation of glycogen synthase kinase 3beta (GSK3beta), a negative regulator of cardiac hypertrophy. Therefore, we suggested that CHP3 is one of the negative regulators of cardiomyocyte hypertrophy. In this study, we focused on the role of CHP3 in skeletal muscle. In mouse myoblast cell line, C2C12, almost no endogenous CHP3 protein was expressed. However, the expression level was increased in differentiated myotubes. Immunofluorescent studies showed that CHP3 was co-localized with GSK3beta in the myotube. We will discuss the role of CHP3 in mouse skeletal muscle.