Intestinal epithelial cells constitute the first line of defence against the entry of toxins and pathogens from the lumen to the body, with several processes contributing to this innate barrier function. For example, fluid secretion flushes pathogens from the lumen, defensin production protects against bacterial invasion, autophagy aids in clearing bacteria from infected cells, and restitution repairs the epithelium after injury. Although bile acids are known to be involved in the pathogenesis of IBD, the mechanisms by which they regulate epithelial barrier function are not well understood. Therefore, the aim of this study was to investigate the effects of deoxycholic acid (DCA) on epithelial responses that contribute to barrier function. T84colonic adenocarcinoma cells were grown on permeable supports and were treated with DCA (50 µM -1 mM) for 1 h to 72 h. Protein expression was investigated by western blotting and mRNA expression by qPCR. Epithelial restitution in polarised T 84 cell monolayers was investigated using a wound healing assay. Muscle stripped sections of human colonic mucosa were mounted in Ussing chambers for measurements of ion transport by monitoring changes in short circuit current (Isc) under voltage-clamped conditions. All experiments involving human tissue had the approval of Beaumont Hospital Ethical Committee. DCA (150 µM, 48 h) attenuated restitution of mechanically wounded T 84 cell monolayers by 2.08 ± 0.14 fold compared to untreated controls (n = 3; p < 0.05). Furthermore, DCA (200 µM, 24 h) stimulated autophagy in T84 cells, as indicated by a 1.8 ± 0.9 fold increase in the expression of the autophagy marker, microtubule-associated protein light chain 3 (LC-3 II), when compared to controls (n = 5; p < 0.001). In contrast, the more hydrophillic bile acid, UDCA (50-500 µM) was without effect on LC-3 II expression. DCA also increased expression of mRNA for the constitutively expressed hβD-1 by 7.0 ± 0.9 fold, compared to untreated cells (n = 5; p < 0.001). Levels of inducible hβD-2 mRNA were also significantly upregulated by 4.3 ±0.4 fold in response to DCA (n = 4; p < 0.01). When added to voltage clamped sections of human colonic tissue, DCA, at concentrations of 200 µM and 500 µM, stimulated Isc responses of 4.0 ± 0.0 µA/cm2 and 24.0 ± 0.0 µA/cm2 (n = 2). Furthermore, DCA pretreatment attenuated Cl- secretory responses to the Ca2+ dependent agonist, carbachol (CCh, 100 µM) from 320 ± 274 µA/cm2 to 56 ± 3 µA/cm2, n = 2). At concentrations of > 500 µM, DCA rapidly damaged colonic tissues, as demonstrated by a 5.7 ± 0.9 fold increase in transmucosal conductance (n = 2). Taken together, these data implicate colonic bile acids, such as DCA, as important regulators of intestinal epithelial barrier function and suggest they might be good targets for the development of new approaches to treat IBD.