Epithelial Cl- secretion, the main driving force for intestinal fluid secretion, can become dysregulated in conditions of disease, leading to diarrhoea. We have previously shown that agonists of the nuclear bile acid receptor, farnesoid X receptor (FXR), prevent secretory diarrhoea and that this effect is accompanied by decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Here, we set out to further investigate the role of bile acids and the FXR in regulation of CFTR expression.The naturally-occurring bile acids, deoxycholic (DCA) and chenodeoxycholic (CDCA), or synthetic FXR receptor agonists, GW4064 and obeticholic acid (OCA), were used to activate FXR. mRNA expression was measured by qPCR, while protein expression by immunoblotting. To measure CFTR promoter activity, HEK293 cells were transfected with plasmids expressing FXR, retinoid X receptor (RXR) and the CFTR gene promoter linked to a luciferase reporter. First, we examined the effects of FXR activation on CFTR mRNA expression and found that in GW4064-treated (5 μM; 24 hrs) T84 colonic epithelial cells, CFTR mRNA levels were decreased to 47 ± 13% of that in controls (n=5, p<0.05). Bioinformatic analysis of the CFTR promoter sequence revealed the presence of several putative FXR responsive elements. To determine if decreases in CFTR mRNA levels were due to repression of CFTR promoter activity, we carried out luciferase reporter assays. In GW4064- treated HEK293 cells, CFTR promoter activity was reduced to 29 ± 3% of that in controls (n=4, p<0.001), indicating FXR negatively regulates the CFTR promoter. Next, we investigated if naturally-occurring agonists of the FXR, DCA and CDCA, also inhibit CFTR. DCA (50 μM) induced nuclear translocation of FXR in T84 cells, and reduced CFTR protein expression to 19 ± 9% of that in controls (n=3, p < 0.05). DCA also repressed CFTR promoter activity in HEK293 cells to 38 ± 5% (n=3, p<0.001) of controls. In cells that were not transfected with FXR, effects of DCA on CFTR promoter activity were not apparent (n = 3). CDCA (50 μM) and OCA (50 μM), were also found to inhibit CFTR promoter activity to 20 ± 1% (n=3, p<0.001) and 20 ± 2% of controls (n=3, p<0.001), respectively. In contrast, the conjugated bile acid, TDCA (100 μM), was without effect on CFTR promoter activity (n=3).In conclusion, we have discovered a new role for bile acids as repressors of epithelial CFTR expression, an effect which is mediated by FXR-induced inhibition of CFTR promoter activity. These findings have important implications for our understanding of intestinal transport regulation by bile acids and suggest that FXR agonists may be useful agents in treating diarrheal diseases.