Intestinal epithelial cells compose the first line of innate defence in colon. For example, fluid secretion flushes pathogens from the lumen, defensins protect against bacterial invasion and restitution repairs the epithelium after injury. Although bile acids are also known to be involved in the pathogenesis of Inflammatory Bowel Disease (IBD), the mechanisms behind their actions are not well understood. The aim of this study was to investigate the effects of the colonic bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on innate epithelial barrier function. T84 colonic adenocarcinoma cells were grown on permeable supports and were treated with DCA and UDCA (10 µM – 1mM) for 1 – 72 h. Protein secretion was investigated by ELISA. Epithelial restitution in polarised monolayers was evaluated by a wound healing assay. Muscle stripped sections of human colonic mucosa were mounted in Ussing chambers for measurements of β-defensin secretion and ion transport by monitoring changes in short circuit current (Isc) under voltage-clamped conditions. Data were statistically analysed by Student t-tests or two-way ANOVA with the Newman-Keuls multiple comparison post-test. Experiments involving human tissue were approved by the Beaumont Hospital Ethics Committee. When added to human colonic tissue, DCA, at concentrations ≥ 200 µM, stimulated Isc responses of 11 ± 4 µA/cm2 (p < 0.05; n = 4). Furthermore, DCA pretreatment attenuated Cl- secretory responses to the Ca2+-dependent agonist, carbachol (CCh, 100 µM) from 219 ± 71 µA/cm2 to 115 ± 26 µA/cm2 (n = 4). In T84 monolayers, DCA stimulated secretion of both human β defensin-1 (HβD-1) and HβD-2 into the apical media. A specific inhibitor of the NF-κB pathway, BMS-34451, significantly attenuated DCA (150 µM)-stimulated HβD-2 secretion from 115 ± 8 pg/ml to 27 ± 8 pg/ml (n = 6; p < 0.01). In contrast, UDCA (50 µM) reduced basal HβD secretion and attenuated DCA induced HβD-1 release from 683 ± 131 pg/ml to 304 ± 94 pg/ml and HβD-2 release from 200 ± 42 pg/ml to 21 ± 11 pg/ml (n = 8; p < 0.001). A specific agonist of the TGR-5 receptor, INT777, mimicked the effects of DCA on HβD-1 and HβD-2 secretion and UDCA attenuated TGR-5-induced secretion of HβD-1 from 429 ± 95 pg/ml to 215 ± 65 pg/ml and HβD-2 from 105 ± 23 pg/ml to 35 ± 3 pg/ml (n = 6: p < 0.01).Finally, DCA prevented and UDCA promoted restitution of wounded epithelial cell monolayers. Furthermore, co-treatment of the cells with UDCA (100 µM, 48 h) reversed the effects of DCA. In DCA-treated cells, the extent of wound closure was 29 ± 15 % after 48 h, compared to 79 ± 5 % in cells co-treated with UDCA (n = 5; p < 0.001). Taken together, these data implicate the colonic bile acids, DCA and UDCA, as important regulators of innate epithelial barrier function in the intestine, and suggest that bile acids are good targets for the development of new approaches to treat IBD.