Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating GSK3 through phosphorylation. Insulin also promotes glucose uptake and glucose-6-phosphate (G6P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphoryltion of GS mediated via GSK3 is required for normal glycogen synthesis in muscle with insulin. We employed GSK3 knock-in mice in which wild-type GSK3 and genes are replaced with mutant forms (GSK3|/^{S21A/S21A/S9A/S9A}), which are non-responsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3/^{S21A/S21A/S9A/S9A} mice, glycogen content in different muscles from these mice was similar compared to wild-type mice. Basal and adrenaline-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knock-in mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5mM glucose in the presence or absence of insulin revealed that the levels of G6P, the rate of [¹⁴C]glucose incorporation into glycogen, as well as an increase in total glycogen content were similar between wild-type and GSK3 knock-in mice. Injection of glucose containing [³H]2-deoxy-glucose and [¹⁴C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knock-in mice. These results suggest that insulin-mediated inhibition of GSK3 is not a rate-limiting step in muscle glycogen synthesis in mice. This suggests that allosteric regulation of GS by G6P may play a key role in insulin-stimulated muscle glycogen synthesis in vivo.