Epithelial restitution is an essential process for maintenance of intestinal barrier function. Increased levels of colonic bile acids have been proposed to be involved in the pathogenesis of inflammatory bowel disease (IBD) but their roles in regulating restitution are not yet known. Here, we investigated the effects of bile acids on epithelial restitution in a model of mechanically wounded colonic epithelial cells. T84 colonic epithelial cells, grown as monolayers on transparent permeable supports, were wounded by scratching with a pipette tip at T=0. Cells were treated with either deoxycholic acid (DCA; 150 μM) or ursodeoxycholic acid (UDCA; 100 µM) and restitution was measured as wound area after 48 h expressed as % T=0values. After 48 h, wounds spontaneously closed to 37 ± 13% of T=0 values. In the presence of DCA, wound healing was reduced (wound size = 76 ± 13%), whereas UDCA enhanced healing (wound size = 12 ± 2%) (n = 5; p < 0.001). Furthermore, UDCA completely prevented inhibition of wound closure by DCA. The cystic fibrosis transmembrane conductance regulator, CFTR, has been previously shown to be important in progression of wound healing in epithelial cells. We found that DCA decreased expression of CFTR Cl- channels to 60 ± 10% of that in control cells, while a CFTR inhibitor, CFTR(inh)-172 (10 μM), attenuated wound closure (wound size = 63 ± 2%) (n = 6; p < 0.01). CFTR(inh)-172 inhibition of wound closure was also prevented by UDCA. Finally, GW4064 (5 µM), an agonist of the nuclear bile acid receptor, FXR, mimicked DCA effects on wound healing and CFTR expression. Our data suggest that colonic bile acids differentially regulate intestinal epithelial restitution. DCA prevents wound healing by a mechanism that likely involves FXR-mediated downregulation of CFTR expression, while UDCA promotes healing and protects against the detrimental effects of DCA. Thus, therapeutic manipulation of the colonic bile acid pool may prove to be a useful approach for promoting intestinal barrier function in IBD.