Glucokinase (GK) plays a key role in the regulation of glucose utilization and glucose stimulated insulin secretion in pancreatic islet cells. Gene-targeting of the insulin-like growth factor I receptor (IGF-IR) down regulated pancreatic islet GK activity. That finding prompted us to examine potential mechanism that may control GK gene activity using an islet cell line, INS-1 known to express IGF-IR. Exposure of these cells to IGF-I induced GK protein expression and activity of the enzyme in a dose-dependent manner. In addition, IGF-I induced activity of a reporter construct containing the GK promoter in parallel with the effect on endogenous GK mRNA levels. The stimulatory effect of IGF-I on GK promoter activity was abrogated by wortmannin, and LY294002, specific inhibitors of PI3-K. Exposure of cells to IGF-I elicited a rapid phosphorylation of Akt and FoxO1, a known target of Akt signaling. Constitutively active Akt stimulates the activity of the GK promoter and a dominant-negative mutant of Akt or mutagenesis of a FoxO1 response element in the GK promoter abolished the ability of IGF-I to stimulate the promoter activity. Furthermore, cell knock-down of FoxO1 with siRNA disrupted the effect of IGF-I on GK expression. These results demonstrate that PI3-K/Akt/FoxO1 pathway contribute to the regulation of GK gene expression in response to IGF-I stimulation.