Phosphoinositide 3-kinase (PI3K) p85-deficient mice exhibited hypoglycemia as a result of increased insulin sensitivity and glucose uptake in peripheral tissues. Although PI3K is central to the metabolic actions of insulin, its mechanism of action in liver is not well understood. In the present study, we investigated hepatic insulin signaling and glucose homeostasis in p85-deficient and wild-type mice. In the livers of p85-deficient mice, p50 played a compensatory role in insulin-stimulated PI3K activation by binding to IRS-1/2. In p85-deficient mice, the ratio of p50 over p110 catalytic subunit of PI3K in the liver was higher than in the muscles. PI3K activity associated with IRS-1/2 was not affected by the lack of p85 in the lever. Insulin-stimulated Akt and PTEN activities in the liver were similar in p85-deficient and wild-type mice. Hyperinsulinemic-euglycemic clamp study revealed that the glucose infusion rate and the rate of disappearance were higher in p85-deficient mice than in wild-type mice, but endogenous glucose production tended to be higher in p85-deficient mice than in wild-type mice. Consistent with this finding, the expressions of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in livers after fasting were higher in p85-deficient mice than in wild-type mice. After fasting, the intrahepatic glucose-6-phosphate level was almost completely depleted in p85-deficient mice. The glycogen content fell to nearly zero as a result of glycogenolysis shortly after the initiation of fasting in p85-deficient mice. The absence of an increase in insulin-stimulated PI3K activation in the liver of p85-deficient mice, unlike the muscles, may be associated with the molecular balance between the regulatory subunit and the catalytic subunit of PI3K. Gluconeogenesis was rather elevated in p85-deficient mice, compared with in wild-type mice, and the liver seemed to partially compensate for the increase in glucose uptake in peripheral tissues.