Cl- secretion, the main driving force for intestinal fluid secretion, can become dysregulated in conditions of disease, leading to the onset of diarrhoea. While such diseases, which include irritable bowel syndrome and infectious diarrhoeas, constitute a huge health and economic burden to society, currently available medications are unsatisfactory and new, more specific, therapies are required. Although we have previously shown that, at physiological concentrations, colonic bile acids prevent epithelial Cl- secretion, the mechanisms involved remain unclear. Here we investigated a potential role for the nuclear bile acid receptor, farnesoid X receptor (FXR), in regulation of intestinal secretory responses and as a new target for development of antidiarrhoeal drugs. GW4064 (5 μM for 24 hrs) was used to activate the FXR. Cl- secretion was measured as changes in short-circuit current across T84 cell monolayers or muscle-stripped mouse colonic tissues in Ussing chambers. Protein expression was measured by western blotting. GW4064 treatment activated the FXR and inhibited responses to the Ca2+- and cAMP-dependent secretagogues, carbachol (CCh) and forskolin (FSK), to 56 ± 5 (p < 0.001) and 60 ± 5% (p < 0.001) of controls, respectively (n=15). GW4064 (10 μM) also inhibited responses to the naturally-occurring secretagogues, cholera toxin (0.1 μg/ml) and deoxycholic acid (500 μM) to 81 ± 6 (n=7, p < 0.05) and 64 ± 10% (n=10, p < 0.05) of controls, respectively. Similar to its effects in cultured epithelia, intraperitoneal injection of GW4064 to mice (50 mg/kg; n=11) attenuated CCh- and FSK-stimulated secretory responses in colonic tissues. However, jejunal sodium-dependent glucose co-transport and colonic ENaC currents were not decreased, suggesting the effects of FXR activation to be specific for secretory processes. Furthermore, GW4064 treatment dramatically inhibited the severity of symptoms in a mouse model of diarrhoeal disease (n=10). In experiments to determine molecular mechanisms involved, we found that GW4064 decreased CFTR-mediated Cl- currents in T84 cells to 68 ± 5% (n=9; p < 0.01) of control values, and this was associated with a 33 ± 9% decrease in CFTR protein expression (n=4; p < 0.01). GW4064 also inhibited Na+/K+-ATPase activity to 61 ± 10% of controls (n=6; p < 0.05). However, while GW4064 did not alter expression of the Na+/K+-ATPase, it did increase expression of a regulatory protein, FXYD3, to 166 ± 16% (n=8, p < 0.01) of control values. These data reveal novel antidiarrhoeal actions of FXR in colonic epithelial cells. These effects are mediated by direct inhibition of multiple components of the Cl- secretory pathway, without altering key absorptive processes. Our data suggest that FXR agonists may be useful in treating secretory diarrhoeas associated with common intestinal disorders.