Glycogen synthase kinase (GSK)-3β inhibits glycogen storage, protein synthesis, and has recently been shown to inhibit the calcineurin-dependent transcriptional effects of nuclear factor of activated T-cells (NFAT) by re-phosphorylation and subsequent exclusion from the nucleus. We hypothesized that calcium may facilitate adaptive responses in skeletal muscle cells by inhibiting GSK-3β. Further, this effect may be mediated by the signaling molecule nitric oxide (NO). Methods: C2C12 cells were seeded on 6 well plates and incubated in 10% Fetal Bovine Serum and changed to 2% Horse Serum at 5 days to induce differentiation. Myotubes were grown to 80% confluency and were treated with calcium ionophore A23187, L-NAME, A23187 + L-NAME, or PAPA-NO. Protein lysates were separated by SDS-PAGE and quantified by immunoblot. Results: GSK-3β phosphorylation status (p-GSK/total GSK) was increased by A23187 in a dose-dependent manner, indicating an inhibiting effect. L-NAME treatment alone did not affect baseline phosphorylation of GSK-3β. However, co-treatment with L-NAME completely inhibited the A23187-induced phosphorylation of GSK-3β, demonstrating the necessity of NO for the A23187 effect. Further, PAPA-NO treated cells increased p-GSK/total GSK by 50% compared to control. GSK-3β inhibition via calcium is, therefore, dependent on NO levels in myocytes.