Background: Cl^– secretion is the primary driving force for fluid secretion in the intestine. In patients with bile acid malabsorption, pathophysiologically high (mM) concentrations of dihydroxy bile acids enter the colon and stimulate Cl^– secretion, thereby causing diarrhea. However, under normal physiological circumstances, deoxycholic acid (DCA) is the predominant luminal bile acid and is present at lower (µM) concentrations; its role in regulating secretion under such conditions is unclear. Thus, the aim of this study was to investigate the effects of physiological DCA concentrations on colonic epithelial secretory function. Methods: Cl^– secretion was measured as changes in short circuit current (I_SC) across voltage-clamped monolayers of T84 colonic epithelial cells in Ussing chambers. Protein expression was measured by Western blotting. Results: Acute exposure to high concentrations of DCA (1 mM) rapidly stimulated Cl^– secretion across T84 cells; an effect that was associated with epithelial injury as evidenced by decreases in transepithelial resistance (TER) and increased lactate dehydrogenase (LDH) release. Acute exposure to low concentrations (100μM) of DCA did not alter basal Cl^– secretion, however prolonged exposure (24hrs) inhibited secretory responses to both the Ca²⁺– and the cAMP- dependent agonists, carbachol (CCh; 100 µM) and forskolin (FSK; 10 µM) to 39.4 ± 7.7% (n=10; p<0.001) and 73.3 ± 5.9% (n=5; p<0.01) of those in control cells, respectively. Under such conditions, DCA did not alter TER or LDH release. This chronic antisecretory effect of DCA occurred with an EC₅₀ of 75 µM, was apparent within 3 hrs and was maximal by 6 hrs after exposure to the bile acid. Further investigation revealed that DCA did not alter CCh-induced mobilization of intracellular calcium, implying its antisecretory actions are not due to alterations in the generation of 2nd messengers. Chronic exposure to DCA did not alter the expression of NKCC1 or the Na⁺/K⁺ ATPase pump at the protein level, nor did it alter apical Cl^– channel or basolateral K⁺ channel conductances. However, the antisecretory effects of DCA were blocked by the inhibitor of protein translation, cycloheximide. Conclusion: By virtue of its ability to stimulate secretion at high concentrations and to inhibit secretion at low concentrations, we propose a novel role for DCA as a colonic “osmosignal” that regulates the fluidity of the luminal contents. The antisecretory effects of DCA are mediated by de novo protein synthesis but do not appear to involve alterations in the activity or expression of key transport proteins of the Cl^– secretory mechanism.