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, IGF-I was applied to differentiating C2C12 myoblasts, and the effects on phosphatidyl-inositol 3-kinase (PI-(3)K)/Akt /Glycogen Synthase Kinase 3 (GSK-3) signaling and myogenesis were evaluated. IGF-I caused phosphorylation and inactivation of GSK-3 activity via signaling through the PI-(3)K/Akt pathway. We assessed whether pharmacological inhibition of GSK-3 using 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, mRNA's 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-3 inhibition abrogated IGF-I, but not LiCl dependent stimulation of myogenic mRNA accumulation, suggesting that the pro-myogenic effects of IGF-I require GSK-3 inactivation, and revealing an important negative regulatory role for GSK-3 in myogenesis. Therefore, this study identifies GSK-3 as a potential target for pharmacological stimulation of muscle growth.