Nutrition availability is one of the major environmental signals influencing cell fates such as proliferation, differentiation and apoptosis, often functioning in concert with other humoral factors including insulin. Herein, we show that low serum-induced differentiation of C2C12 myocytes is significantly hampered under low glucose (LG: 5 mM glucose) as compared with high glucose (HG: 22.5 mM glucose) conditions, concurrently with nuclear accumulation of SIRT1, an NAD⁺-dependent deacetylase, and FoxO3a, both of which are implicated in the negative regulation of myogenesis. Intriguingly, insulin appears to exert opposite actions, depending upon glucose availability, in regard to the regulation of SIRT1 and FoxO3a abundance, which apparently contributes to modulating the potency of insulin's myogenic action. Namely, insulin exerts a potent myogenic effect in the presence of sufficient levels of glucose, while insulin is unable to exert its myogenic action under LG conditions, since insulin evokes massive up-regulation of both SIRT1 and FoxO3a in the absence of sufficient ambient glucose. In addition, the retarded differentiation state under LG is significantly restored by sirtinol, a SIRT1 inhibitor, whereas insulin abolished this sirtinol-dependent restoration, indicating that insulin can function as a negative, as well as a positive, myogenic factor depending upon glucose availability. Together, our data reveal the importance of ambient glucose levels in the regulation of myogenesis and also in the determination of insulin's myogenic potency, which is achieved at least in part through regulation of the cellular contents and localization of SIRT1 and FoxO3a in differentiating C2C12 myocytes.