Hepatic insulin resistance is one of the characteristics of type 2 diabetes and contributes to the development of hyperglycemia. How changes in hepatic glucose flux lead to insulin resistance is not clearly defined. We determined the effects of decreasing the levels of hepatic fructose-2,6-bisphosphate on hepatic glucose flux in the normal 129J mice. Upon adenoviral overexpression of a kinase activity-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme that determines fructose-2,6-bisphosphate level, hepatic fructose-2,6-bisphosphate levels were decreased 2-fold compared to those of control virus-treated mice in basal state. In addition, under hyperinsulinemic conditions, hepatic fructose-2, 6-bisphosphate levels were much lower than those of the control. The decrease in F26P2 leads to the elevation of basal and insulin-suppressed hepatic glucose production. Also, the efficiency of insulin to suppress hepatic glucose production was decreased (63.3%-suppression vs. 95.5%-suppression of the control). At the molecular level, a decrease in insulin-stimulated Akt phosphorylation was consistent with hepatic insulin resistance. In the low hepatic fructose-2,6-bisphosphate-states, increases in both gluconeogenesis and glycogenolysis in the liver are responsible for elevations of HGP, and thereby contribute to the development of hyperglycemia. Additionally, the increased hepatic gluconeogenesis was associated with the elevated mRNA levels of peroxisome proliferator activated receptor- co-activator 1 and phosphoenolpyruvate carboxykinase. This study provides the first in vivo demonstration that decreasing hepatic fructose-2,6-bisphosphate levels leads to increased gluconeogenesis in the liver. Taken together, the current study demonstrates that perturbation of glucose flux in the liver play a predominant role in the development of a diabetic phenotype as characterized by hepatic insulin resistance.