Intestinal epithelia constitute the first line of defence with several processes contributing to this innate barrier function. For example, fluid secretion flushes pathogens from the lumen, defensin protects against bacterial invasion and restitution repairs the epithelium after injury. Although bile acids are known to be involved in the pathogenesis of Inflammatory Bowel Disease (IBD), the regulatory mechanisms are not well understood. The aim of this study was to investigate the effects of deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) on epithelial responses that contribute to barrier function.T84 colonic adenocarcinoma cells were grown on permeable supports and were treated with DCA (10 µM – 1 mM) and UDCA (10 – 300 µM) for 1 – 72 h. Protein expression was investigated by western blotting, protein secretion by ELISA and mRNA expression by qPCR. Epithelial restitution in polarised T84 cell monolayers was investigated using 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 using a Student T-test or Two-way ANOVA with Newman-Keuls Multiple Comparison Post Test. All experiments involving human tissue had the approval of Beaumont Hospital Ethical Committee.When added to voltage clamped sections of 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 cells, UDCA (50 µM) attenuated DCA (150 µM) stimulated human β defensin-1 (HβD-1) release from 683 ± 131 pg/ml to 304 ± 94 pg/ml and human β defensin-2 (HβD-2) release from 200 ± 42 pg/ml to 21 ± 11 pg/ml (n = 8; p < 0.001). Using a specific agonist of TGR-5 receptor (INT777), we determinated that HβD-1 and HβD-2 are TGR-5 activation dependent and UDCA attenuates TGR-5 activated 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). Using specific inhibitor of NF-κB pathway (BMS-34451), we determined that NF-κB inhibition attenuated DCA (150 µM) stimulated HβD-2 secretion from 115 ± 8 pg/ml to 27 ± 8 pg/ml (n = 6; p < 0.01). Moreover UDCA (100 µM, 48 h) showed more of its protective properties by preventing DCA (150 µM) induced inhibition of epithelial restitution of mechanically wounded cell monolayers from 29 ± 15 % to 79 ± 5 % (n = 5; < 0.001).Taken together, these data implicate colonic bile acids, DCA and UDCA, as important regulators of intestinal epithelial barrier function and suggest they are promising targets for the development of new approaches to treat IBD.