Bile acids regulate the expression of genes involved in cholesterol homeostasis. They are ligands of the farnesoid X receptor, which induces small heterodimer partner (SHP)-1, a transcriptional repressor of bile acid synthetic enzymes. In cholestatic liver disease, hepatic bile acid concentrations are elevated and expression of the major Na+−independent bile acid uptake system, organic anion transporting polypeptide (OATP)-C (solute carrier gene family SLC21A6), is markedly decreased. Because the OATP-C gene is transcriptionally dependent on the hepatocyte nuclear factor (HNF) 1α, we hypothesized that bile acids decrease OATP-C expression through direct repression of HNF1α. To test this hypothesis, we studied the regulation of the human HNF1α gene by bile acids. HNF1α expression in cultured hepatoma cells was decreased ∼50% after 12 hours' exposure to 100 μmol/L chenodeoxycholic acid (CDCA). Characterization of the human HNF1α gene promoter identified a consensus bile acid response element that binds and is activated by HNF4α. Mutagenesis of the HNF4α site abolished baseline HNF1α promoter activity. The central mechanism by which bile acids repress HNF1α is decreased activation by HNF4α. SHP directly inhibits HNF4α-mediated transactivation of the HNF1α promoter in cotransfection assays. In addition, HNF4α nuclear binding activity is decreased by CDCA and the human HNF4α gene promoter is repressed by CDCA through an SHP-independent mechanism. In conclusion, we show that repression of HNF1α is an important new mechanism by which bile acids regulate the expression of HNF1α-dependent genes in man. This explains the suppressive effect of bile acids on the OATP-C gene promoter, leading to decreased expression in cholestatic liver disease.