Abnormal hepatic gluconeogenesis contributes significantly to both fasting and non-fasting hyperglycemia of patients with type 2 diabetes. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates the key hepatic gluconeogenic enzymes including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) through the amplification of glucocorticoid receptor (GR) - mediated tissue glucocorticoid action, and is crucially dependent on hexose-6-phosphate dehydrogenase (H6PDH) - generating NADPH system. Here, we observed that compared with fasting state, H6PDH and 11β-HSD1 expression in livers were all increased under non-fasting state in both normal and diabetic rats, and the non-fasting diabetic group was the highest among the four experimental groups. Moreover, incubation of primary hepatocytes with increasing glucose caused dose-dependent increases in H6PDH, 11β-HSD1, GR, PEPCK and G6Pase expression. Also, glucose-6-phosphate (G6P) had a positive regulation on H6PDH and 11β-HSD1 in hepatocytes. In addition, primary hepatocytes treated with different doses of insulin in high glucose induced alteration of H6PDH and 11β-HSD1 while in low glucose there was no significant effect. These findings suggest that glucose instead of insulin directly regulates H6PDH and 11β-HSD1 and suppression of the two enzymes could be considered as an effective target for the treatment of type 2 diabetes. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.