Background NF-κB p65 was shown to inhibit transcription of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis in the liver. To understand the mechanism of action of NF-κB p65, we investigated the nuclear receptor corepressor in the regulation of PEPCK transcription.Methods Rat H4IIE cells, human hepatoma HepG2 cells and human embryo kidney (HEX) 293 cells were used in this study. The transcriptional activity of a rat PEPCK gene promoter (-490/+100) was analyzed in HepG2 cells, a HepG2 super suppressor IkBα (sslkBα) stable cell line, and HEK 293 cells. The effects of p65 and sslkBa on a rat PEPCK gene promoter were observed using the PEPCK luciferase reporter system. The interaction of the cAMP-response-element-binding (CREB) protein, histone deacetylase 3 (HDAC3) and silencing mediator for retinoic and thyroid hormone receptors (SMRT) with the PEPCK gene promoter were investigated using the chromatin immunoprecipitation (ChIP) assay. p65 cotransfection and RNAi-mediated gene knockdown were used to determine the corepressor involved in the inhibition of PEPCK by NF-κB p65 and the transcriptional regulation of CREB by NF-κB p65.Results NF-κB p65 inhibited PEPCK expression and the inhibition was blocked by sslkBα. The inhibitory effect of p65 was completely blocked in a HepG2 stable cell line in which sslkBα was expressed. HDAC3 or SMRT knockdown led to a significant up-regulation of PEPCK reporter activity in the presence of p65 cotransfection. In the ChIP assay the interaction of HDAC3 and SMRT with the PEPCK gene promoter was induced by p65 activation, but the CREB signal was reduced. Transcriptional activity of CREB was inhibited by NF-κB p65 cotransfection. The inhibitory effect of NF-κB p65 was blocked by HDAC3 RNAi or SMRT RNAi. Conculsions The study showed that the inhibition of PEPCK by NF-κB p65 was dependent on HDAC3 and SMRT, which form a nuclear corepressor complex for transcriptional inhibition. The transcription factors NF-κB p65 and CREB share the same corepressor HDAC3-SMRT, and the corepressor exchange leads to inhibition of PEPCK gene transcription by NF-κB p65.