Intracellular calcium transients in skeletal muscle cells initiate phenotypic adaptations via activation of calcineurin and its effector, nuclear factor of activated t-cells (NFAT). Further, endogenous production of nitric oxide (NO) via calcium-calmodulin-dependent nitric oxide synthase (NOS) is involved in skeletal muscle phenotypic plasticity. Here, we provide evidence that NO enhances calcium-dependent nuclear accumulation and transcriptional activity of NFAT, and induces phosphorylation of GSK-3 in C2C12 myotubes. The calcium ionophore, A23187 (1µM for 9h), or thapsigargin (2µM for 4h) increased NFAT transcriptional activity by 7 and 4-fold, respectively, in myotubes transiently transfected with an NFAT-dependent reporter plasmid (pNFAT-luc, Stratagene). Co-treatment with the NOS-inhibitor, N(G)-L-nitro-arginine methyl ester (L-NAME; 5mM) or the guanylate cyclase inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10µM), prevented the calcium-effects on NFAT activity. The NO donor, diethylenetriamine-NONO (DETA-NO; 10µM) augmented the effects of A23187 on NFAT-dependent transcription. Similarly, A23187 (0.4µM for 4h) caused nuclear accumulation of NFAT and increased phosphorylation (i.e. inactivation) of GSK-3, while co-treatment with L-NAME or ODQ inhibited these responses. Finally, the NO donor, 3-(2-Hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA-NO; 1µM for 1h) increased phosphorylation of GSK-3 in a manner dependent upon guanylate cyclase activity. We conclude that NOS activity mediates calcium-induced phosphorylation of GSK-3 and activation of NFAT-dependent transcription in myotubes. Furthermore, these effects of NO are guanylate cyclase-dependent.