Inhibition of phosphoenolpyruvate carboxykinase (PEPCK) by tumor necrosis factor (TNF-) contributes to the pathogenesis of hypoglycemia in endotoxin shock. In this study, the molecular mechanism underlying the inhibition was investigated in hepatoma cells (rat H4IIE and human HepG2). PEPCK expression was induced by cAMP and the induction was reduced by TNF-a at protein and mRNA levels in H4IIE cells. The inhibition was observed in the PEPCK gene promoter in a PEPCK-Luc reporter. Activation of NF-kB pathway was required for the transcriptional inhibition of PEPCK gene. IkBa degradation and p65 nuclear translocation were involved in the inhibition. An interaction of HDAC3 and SMRT with the PEPCK gene promoter was induced by TNF- and observed in a ChIP assay. The TNF-induced inhibition was blocked by HDAC inhibitor or HDAC3 knockdown. The blocking effect was also observed in knockdown of corepressor SMRT. Point mutation suggests that CRE element is required for TNF-induced inhibition of the PEPCK gene promoter. CREB phosphorylation at Ser133 and expression of PGC-1 were not changed by TNF- in H4IIE cells. The transcriptional activity of CREB was inhibited by TNF- in a CRE-Luc reporter. The data suggests that the nuclear corepressor proteins of HDAC3 and SMRT mediate TNF-inhibition of PEPCK transcription. The inhibition mechanism is related to activation of NF-kB and inhibition of CREB activity by the corepressor. These data suggest a novel activity of nuclear corepressor in the regulation of PEPCK expression by TNF-.