The liver responds to an increase in blood glucose levels in the postprandial state by uptake of glucose and conversion to glycogen. Liver glycogen synthase (GYS2), a key enzyme in glycogen synthesis, is controlled by a complex interplay between the allosteric activator glucose-6-phosphate (G6P) and reversible phosphorylation through GS kinase-3 and glycogen-associated form of protein phosphatase 1. Here we initially performed mutagenesis analysis and identified a key residue (Arg582) required for activation of GYS2 by G6P. We then employed GYS2 Arg582Ala knockin (+/R582A) mice in which G6P-mediated GYS2 activation has been profoundly impaired (60-70%), while sparing regulation through reversible phosphorylation. R582A-mutant-expressing hepatocytes showed significantly reduced glycogen synthesis with glucose and insulin or glucokinase activator, which resulted in channeling glucose/G6P towards glycolysis and lipid synthesis. GYS2(+/R582A) mice were modestly glucose intolerant and displayed significantly reduced glycogen accumulation with feeding or glucose load in vivo. These data show that G6P-mediated activation of GYS2 plays a key role in controlling glycogen synthesis and hepatic glucose-G6P flux control and thus whole-body glucose homeostasis.