Skeletal muscle atrophy is a prominent and disabling feature of chronic wasting diseases. Prevention or reversal of muscle atrophy by administration of skeletal muscle growth (hypertrophy)-stimulating agents such as insulin-like growth factor I (IGF-I) could be an important therapeutic strategy in these diseases. To elucidate the IGF-I signal transduction responsible for muscle formation (myogenesis) during muscle growth and regeneration, we applied IGF-I to differentiating C(2)C(12) myoblasts and evaluated the effects on phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase-3beta (GSK-3beta) signaling and myogenesis. IGF-I caused phosphorylation and inactivation of GSK-3beta activity via signaling through the PI3K/Akt pathway. We assessed whether pharmacological inhibition of GSK-3beta with lithium chloride (LiCl) was sufficient to stimulate myogenesis. Addition of IGF-I or LiCl stimulated myogenesis, evidenced by increased myotube formation, muscle creatine kinase (MCK) activity, and troponin I (TnI) promoter transactivation during differentiation. Moreover, mRNAs encoding MyoD, Myf-5, myogenin, TnI-slow, TnI-fast, MCK, and myoglobin were upregulated in myoblasts differentiated in the presence of IGF-I or LiCl. Importantly, blockade of GSK-3beta inhibition abrogated IGF-I- but not LiCl-dependent stimulation of myogenic mRNA accumulation, suggesting that the promyogenic effects of IGF-I require GSK-3beta inactivation and revealing an important negative regulatory role for GSK-3beta in myogenesis. Therefore, this study identifies GSK-3beta as a potential target for pharmacological stimulation of muscle growth.