Skeletal muscle atrophy is a prominent and disabling feature in many chronic diseases. Prevention or reversal of muscle atrophy by stimulation of skeletal muscle growth could be an important therapeutic strategy. Glycogen synthase kinase 3beta (GSK-3beta) has been implicated in the negative regulation of skeletal muscle growth. Since myogenic differentiation is an essential part of muscle growth, we investigated if inhibition of GSK-3beta is sufficient to stimulate myogenic differentiation and whether this depended on regulation of the transcription factor nuclear factor of activated T-cells (NFAT). In both myogenically converted mouse embryonic fibroblasts and C2C12 myoblasts, deficiency of GSK-3beta protein (activity) resulted in enhanced myotube formation and muscle-specific gene expression during differentiation, which was reversed by reintroduction of wild type but not kinase-inactive (K85R) GSK-3beta. In addition, GSK-3beta inhibition restored myogenic differentiation following calcineurin blockade, which suggested the involvement of NFAT. GSK-3beta-deficient mouse embryonic fibroblasts or myoblasts displayed enhanced nuclear translocation of NFATc3 and elevated NFAT-sensitive promoter transactivation, which was reduced by reintroducing wild type, but not K85R GSK-3beta. Overexpression of NFATc3 increased muscle gene promoter transactivation, which was abolished by co-expression of wild type GSK-3beta. Finally, stimulation of muscle gene expression observed following GSK-3beta inhibition was strongly attenuated in NFATc3-deficient myoblasts, indicating that this response requires NFATc3. Collectively, our data demonstrate negative regulation of myogenic differentiation by GSK-3beta through a transcriptional mechanism that depends on NFATc3. Inhibition of GSK-3beta may be a potential strategy in prevention or treatment of muscle atrophy.