CLIC5 (chloride intracellular channel 5) is a CLIC (chloride intracellular channel) with various functions. Its high expression in skeletal muscle and association with actin-based cytoskeleton suggests that it may play an important role in muscle tissue. This study was conducted to examine whether CLIC5 regulates the proliferation and differentiation of C2C12 myoblasts into myotubes. Differentiation of C2C12 myoblasts induced by switching to a differentiation culture medium was accompanied by a significant increase of CLIC5 protein expression level. Constitutive overexpression of CLIC5 was associated with reduced cell proliferation and more cells from G2/M phase into G0/G1 phase, followed by increased number and size of myotubes and up-regulation of muscle-specific proteins of myosin heavy chain, myogenin and desmin. These results demonstrate that CLIC5 is involved in C2C12 proliferation and myogenic differentiation in vitro.