The transcription factor forkhead boxO1 (FoxO1) is a key mediator in the insulin signaling pathway and controls multiple physiological functions, including hepatic glucose production (HGP) and pancreatic β-cell function. We previously demonstrated that Ser256 in human FOXO1, equivalent to Ser253 in mouse FoxO1, is a key phosphorylation site mediating the effect of insulin as a target of Akt on suppressing FOXO1 activity and expression of target genes responsible for gluconeogenesis. Here, we investigated the role of FoxO1-Ser253 phosphorylation in control of glucose hemostasis in vivo, by generating global FoxO1-S253A/A knock-in (KI) mice, in which FoxO1-Ser253 alleles were replaced with alanine (A substitution), blocking FoxO1-S253 phosphorylation. FoxO1-S253A/A mice displayed mild increases in feeding blood glucose and insulin levels, but decreases in fasting blood glucose and glucagon concentrations, as well as a reduction in the ratio of pancreatic α-cells/β-cells per islet. FoxO1-S253A/A mice exhibited a slight increase in energy expenditure, but barely altered food intake and glucose uptake among tissues. Further analyses revealed that FoxO1-S253A/A enhances FoxO1 nuclear localization and promotes the effect of glucagon on HGP. We conclude that dephosphorylation of S253 in FoxO1 may reflect a molecular basis of pancreatic plasticity during the development of insulin resistance.