Hexose-6-phosphate dehydrogenase (H6PDH) knockout (KO) mice have reduced generation of nicotinamide adenine dinucleotide phosphate (reduced) within the endoplasmic reticulum. As a consequence, 11beta-hydroxysteroid dehydrogenase type 1 enzyme activity switches from a reductase to a dehydrogenase leading to glucocorticoid inactivation. 11beta-Hydroxysteroid dehydrogenase type 1 has emerged as an important factor in regulating hepatic glucose output; therefore, we examined aspects of glucose homeostasis in KO mice. Compared with wild-type mice, KO mice reduced weight gain, displayed peripheral fasting hypoglycemia, improved glucose tolerance, and elevated plasma corticosterone concentrations. Plasma insulin levels in fed and fasted KO mice are normal; however, insulin and plasma glucose levels are reduced 4 h after fasted animals are refed, indicating improved insulin sensitivity. There is preserved induction and activity of the glucocorticoid-responsive gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in fasted KO mice. Glycogen storage is elevated in fed KO liver, with fed glycogenesis rates increased in KO mice. There is normal flux of lactate through gluconeogenesis recovered as plasma glucose, coupled with increased glycogen derived from lactate. These data suggest partial retention of glucocorticoid sensitivity at the level of the liver. We therefore postulate that increased glycogen synthesis may reflect increased flux of glucose-6-phosphate (H6PDH substrate) through to glycogen in the absence of H6PDH mediated metabolism.