Hexose-6-phosphate dehydrogenase (H6PD, EC 1.1.1.47) catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconolactone within the lumen of the endoplasmic reticulum, thereby generating NADPH. NADPH is a necessary cofactor for the reductase activity of 11-hydroxysteroid dehydrogenase type 1 (11-HSD1, EC 1.1.1.146), which converts hormonally inactive cortisone to active cortisol (in rodents, 11-dehydrocorticosterone to corticosterone). Mice with targeted inactivation of H6PD lack 11-HSD1 reductase activity, whereas dehydrogenase activity (corticosterone to 11-dehydrocorticosterone) is increased. We now report that both glucose output and glucose utilization are abnormal in these mice. Mutant mice have fasting hypoglycemia. In mutant primary hepatocytes, glucose output does not increase normally in response to glucagon. Mutant animals have lower hepatic glycogen content when fed and cannot mobilize it normally when fasting. As assessed by RT-PCR, responses of hepatic enzymes to fasting are blunted; enzymes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase) are not appropriately upregulated, and expression of glucokinase, an enzyme required for glycolysis, is not suppressed. Corticosterone has attenuated effects on expression of these enzymes in cultured mutant primary hepatocytes. Mutant mice have increased sensitivity to insulin as assessed by homeostatic model assessment (HOMA) values and by increased glucose uptake by the muscle. The hypothalamic-pituitary-adrenal (HPA) axis is also abnormal. Circulating ACTH, deoxycorticosterone, and corticosterone levels are increased in mutant animals, suggesting decreased negative feedback on the HPA axis. Comparison with other animal models of adrenal insufficiency suggests that many of the observed abnormalities can be explained by blunted intracellular corticosterone actions despite elevated circulating levels of this hormone.