Intestinal fluid movement is driven by osmotic gradients that are, in turn, established by active ion transport across the epithelium. Previously identified as a neuronal bile acid receptor in the gastrointestinal tract, TGR5 has been implicated in the regulation of intestinal motility. It has also been identified as a regulator of chloride (Cl-) secretion across gallbladder epithelium. However, there is still a paucity of information regarding the expression and role of TGR5 on colonic epithelial cells. Aim: To investigate a potential role for TGR5 in regulation of colonic epithelial ion transport. Methods: The semi-synthetic bile acid, 6α-ethyl-23(S)-methylcholic acid (INT-777), was used to activate TGR5, and the muscarinic agonist, carbachol (CCh), was used to promote colonic epithelial Cl- secretory responses. Ion transport was measured as changes in short circuit current (Isc) across muscle-stripped segments of rat colon mounted in Ussing chambers. mRNA expression was measured by RT-PCR. Confocal microscopy was used to assess TGR5 expression and localisation in isolated rat colonic crypts. Results: TGR5 was found to be highly expressed in colonic epithelial cells both at the mRNA and protein levels. Confocal imaging revealed the protein to be localised bilaterally in isolated rat colonic crypts. Acute bilateral addition of INT-777 (100 μM) to voltage-clamped rat colonic mucosa caused a rapid and transient decrease in Isc to 86.9 ± 5.2 % (n = 8, p ≤ 0.05) of initial values, which returned to basal values by 15 minutes. An analysis of the sidedness of the effects of INT-777 revealed it to be effective from the basolateral, but not apical, side. Furthermore, treatment of rat colonic epithelial cells with INT-777 (100 μM) significantly reduced subsequent secretory responses to CCh to 64.15 ± 1.73 % of those in control cells (n = 3, p ≤ 0.01). The effects of TGR5 were not altered by pretreatment of the tissue with the neurotoxin, tetrodotoxin. Conclusions: These studies reveal a novel role for TGR5 in regulating colonic epithelial ion transport. TGR5 activation rapidly reduces basal Isc and inhibits agonist-induced Cl- secretory responses. In contrast to its previously reported actions on gut motility, the effects of TGR5 on colonic epithelial secretory responses appear to be independent of neuronal activation. Our data have important implications for our understanding of how bile acids regulate colonic fluid and electrolyte transport in health and disease and suggest that TGR5 may provide a new target for development of drugs to treat intestinal disorders associated with dysregulated fluid and electrolyte transport.