Insulin inhibits transcription of cholesterol 7 hydroxylase (Cyp7a1), a key gene in bile acid synthesis, and the HNF-4 site in the promoter was identified as a negative insulin response sequence. Using a fasting/feeding protocol in mice and insulin treatment in HepG2 cells, we explored the inhibition mechanisms. Expression of SREBP-1c, an insulin-induced lipogenic factor, inversely correlated with Cyp7a1 expression in mouse liver. Interaction of HNF-4 with its coactivator, PGC-1,, was observed in livers of fasted mice and was reduced after feeding. Conversely, HNF-4 interaction with SREBP-1c was increased after feeding. In vitro studies suggested that SREBP-1c competed with PGC-1 for direct interaction with the AF2 domain of HNF-4. Reporter assays showed that SREBP-1c, but not of a SREBP-1c mutant lacking the HNF-4 interacting domain, inhibited HNF-4/PGC-1 transactivation of Cyp7a1. SREBP-1c also inhibited PGC-1-coactivation of ER, CAR, PXR, and FXR, implying inhibition of HNF-4 by SREBP-1c could extend to other nuclear receptors. In chromatin immunoprecipitation studies, HNF-4 binding to the promoter was not altered, but PGC-1 was dissociated, SREBP-1c and HDAC2 were recruited, and acetylation of histone H3 was decreased upon feeding. Adenovirus-mediated expression of a SREBP-1c dominant negative mutant which blocks the interaction of SREBP-1c and HNF-4, partially but significantly, reversed the inhibition of Cyp7a1 after feeding. Our data show that SREBP-1c functions as a non-DNA binding inhibitor and mediates, in part, suppression of Cyp7a1 by blocking functional interaction of HNF-4 and PGC-1. This mechanism may be relevant to known repression of many other HNF-4 target genes upon feeding.