The effects of fructose-2,6-bisphosphate (F-2,6-P2) on hepatic glucokinase (GK) and glucose-6-phosphatase (G-6-Pase) gene expression were investigated in streptozotocin-treated mice, which exhibited undetectable levels of insulin. Hepatic F-2,6-P2 levels were manipulated by adenovirus-mediated overexpression of 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase. Streptozotocin treatment alone or with infusion of control adenovirus leads to a dramatic decrease in hepatic F-2,6-P2 content compared with normal nondiabetic mice. This is accompanied by a 14-fold decrease in GK and a 3-fold increase in G-6-Pase protein levels, consistent with a diabetic phenotype. Streptozotocin-treated mice that were infused with adenovirus-overexpressing an engineered 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase with high kinase activity and little bisphosphatase activity showed high levels of hepatic F-2,6-P2. Surprisingly, these mice had a 13-fold increase in GK protein and a 2-fold decrease in G-6-Pase protein compared with diabetic controls. The restoration of GK is associated with increases in the phosphorylation of Akt upon increasing hepatic F-2,6-P 2 content. Moreover, the changes in levels of F-2,6-P2 and Akt phosphorylation revealed a pattern similar to that of streptozotocin mice treated with insulin, indicating that increasing hepatic content of F-2,6-P2 mimics the action of insulin. Because G-6-Pase gene expression was down-regulated only after the restoration of euglycemia, the effect of F-2,6-P2 was indirect. Also, the lowering of blood glucose by high F-2,6-P2 was associated with an increase in hepatic nuclear factor 1-α protein, a transcription factor involved in G-6-Pase gene expression. In conclusion, F-2,6-P2 can stimulate hepatic GK gene expression in an insulin-independent manner and can secondarily affect G-6-Pase gene expression by lowering the level of plasma glucose.