The AMP-activated kinase (AMPK) is a serine threonine kinase that functions as a fuel sensor to regulate energy balance at both cellular and whole-body levels. Here we studied how hepatic AMPKα2 isoform affects hepatic glucose production and peripheral glucose uptake in vivo. We generated mice deleted for the AMPKα2 gene specifically in the liver (liverα2KO). Liverα2KO mice were glucose intolerant and hyperglycemic in the fasted state. Hyperglycemia was associated with a 50% higher endogenous glucose production than in controls as assessed in vivo. We then investigated whether this increased glucose production was sensitive to insulin. Insulin, when infused at a rate inducing physiological hyperinsulinemia, totally inhibited endogenous glucose production in liverα2KO mice, showing that they had normal insulin sensitivity. This was confirmed in vivo by normal insulin-induced phosphorylation of Akt and transcriptional regulation of the phosphoenolpyruvate carboxykinase, glucose-6 phosphatase, and pyruvate kinase in liver during the fasted/fed transition. Leptin and adiponectin regulate hepatic glucose production, so we then infused these adipokines into liverα2KO mice. Neither of these adipokines regulated hepatic glucose production in mice lacking hepatic AMPKα2, whereas both did so in control mice. In conclusion, we show that the hepatic AMPKα2 isoform is essential for suppressing hepatic glucose production and maintaining fasting blood glucose levels in the physiological range. We also demonstrate that regulation of hepatic glucose production by leptin and adiponectin, but not insulin, requires hepatic AMPKα2 activity.