The transcription factor NF-κB mediates inflammation and stress signals in cells. To test NF-κB in the control of hepatic insulin sensitivity, we inactivated NF-κB in liver of C57BL/6 mice through inactivation of p65 gene, which was achieved by crossing floxed p65 and Alb-cre mice to generate L-p65-KO mice. KO mice did not exhibit any alterations in growth, reproduction and body weight on a Chow diet. However, the mice exhibited an improvement in systemic insulin sensitivity on a high fat diet (HFD). Hepatic insulin sensitivity was enhanced in the mice as indicated by increased pyruvate tolerance, Akt phosphorylation and decreased gene expression in hepatic gluconeogenesis. In the liver, a decrease in intracellular cAMP was observed with decreased CREB phosphorylation. Cyclic nucleotide phosphodiesterase 3B (PDE3B), a cAMP degrading enzyme, was increased in mRNA and protein. The increase was a result of absence of NF-κB activity. NF-κB was found to inhibit PDE3B transcription through three DNA-binding sites in the gene promoter in response to TNF-α. Body composition, food intake, energy expenditure, systemic and hepatic inflammation were not significantly altered in KO mice on HFD. These data suggest that NF-κB may inhibit hepatic insulin sensitivity by up-regulating cAMP through suppression of PDE3B gene transcription.