Cl- secretion, the driving force for intestinal fluid secretion, is dysregulated in many intestinal disorders leading to the onset of diarrhea, for which limited therapeutic options exist. We have previously shown that activation of the nuclear bile acid receptor, farnesoid X receptor (FXR), potently inhibits epithelial Cl- secretion in vitro. Here we investigate mechanisms involved and the efficacy of FXR agonists in inhibiting epithelial secretion in vivo. GW4064 was used as a prototypical FXR agonist. Cl- secretion was measured as changes in short-circuit current across voltage clamped monolayers of T84 cells or muscle-stripped mouse colon in Ussing chambers. Protein expression was determined by western blotting or cell surface biotinylation. Data are presented as mean ± SEM; Students t-test was used for statistical significance. As previously reported, GW4064 (1 μM) induced nuclear translocation of FXR in T84 cells and inhibited Cl- secretory responses to the Ca2+ and cAMP-dependent agonists, carbachol (CCh) and forskolin (FSK) to 55.6 ± 8.4 and 72.0 ± 4.0% of control values, respectively (n=7; p<0.01). The effect of GW4064 was slow in onset, occurring after 6 hrs and was maximal by 24 hrs. The antisecretory effect of GW4064 (10 μM) was reversed by the transcriptional inhibitor actinomycin D suggesting de novo synthesis of an intermediary protein is involved. Intraperitoneal injection of GW4064 (50 mg/kg) in mice attenuated CCh and FSK-stimulated secretory responses in ex vivo colonic tissue by 31.2% ± 6.9% (n=6; p<0.05) and 34.6% ± 7.6% (n=6, p<0.01), respectively. To determine the molecular target of GW4064, we analyzed the activity of specific transport proteins that comprise the Cl- secretory pathway. While it was without effect on basolateral K+ channel current, GW4064 significantly decreased Na+/K+ ATPase pump activity to 62.1 ± 4.1% of control values (n=5; p<0.01). Western blotting and cell surface biotinylation revealed that inhibition of Na+/K+ ATPase activity was not due to altered abundance or surface expression of the pump. Furthermore, cellular levels of ATP were unaffected by GW4064 treatment. TCTP and FXYD3, intracellular proteins known to modulate Na+/K+ ATPase activity, were found to be expressed in colonic epithelial cells at the mRNA level but neither was altered by GW4064 treatment. FXR agonists exert potent antisecretory actions in vitro and in vivo. These actions are mediated by inhibition of Na+/K+ ATPase activity, without alterations in pump abundance, surface expression, or availability of ATP. Our data suggest that by virtue of their ability to directly target epithelial transport protein activity, FXR agonists may be useful as a new class of drug to treat diarrhoeal disease.