In this study, we investigated the metabolic phenotype of the NF-kappaB p50 knock-out (p50-KO) mice. Compared with wild type mice, the p50-KO mice had an increase in food intake, but a decrease in body fat content. On chow diet, their blood glucose dropped much more than the wild type (WT) mice in the insulin tolerance test. Their glucose infusion rate was 30% higher than that of the WT mice in the hyperinsulinemic-euglycemic clamp. Their hepatic glucose production was suppressed more actively by insulin, and their insulin-induced glucose uptake was not altered in skeletal muscle or adipose tissue. In the liver, their p70S6K (S6K1) protein was significantly lower, and tumor necrosis factor-alpha (TNF-alpha) expression was much higher. Their S6K1 protein was reduced by TNF-alpha treatment in the primary culture of hepatocytes. S6K1 reduction was blocked by the proteasome inhibitor MG132. In their livers, IKK2 (IKKbeta) activity was reduced together with IKKgamma. Their S6K1 degradation was dependent on IKK2 deficiency. Reconstitution of the S6K1 protein in their liver blocked the increase in insulin sensitivity. S6K1 degradation was not observed in hepatocytes of the WT mice. The data suggest that inactivation of NF-kappaB p50 leads to suppression of IKK2 activity in the liver. IKK2 deficiency leads to S6K1 inhibition through TNF-induced protein degradation. The S6K1 reduction may contribute to insulin sensitivity in p50-KO mice. This study suggests that hepatic S6K1 may be a drug target in the treatment of insulin resistance.