Studies have reported a leaky gut barrier in obesity and in animals fed a high-fat diet. Although bile acids are known to have cytotoxic characteristics, their relevance in high-fat-diet-induced barrier dysfunction has not been studied. We studied the potential bile acid -related mechanisms of barrier dysfunction in high-fat-fed mice. Male C57Bl/6J mice were fed on a high-fat or control diet for 15 weeks. Fecal bile acids were analyzed at week 13. Barrier function was measured at the end of the feeding period from duodenum, jejunum, ileum and proximal colon in an Ussing chamber system using 4 kDa FITC-labeled dextrans. The effects and interaction of deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) on barrier function, epithelial tight junction integrity and inflammation were assayed in vitro. High-fat feeding increased permeability in jejunum (p=0.03) and proximal colon (p=0.01, N=10-14 per group). The proportion of UDCA in fecal bile acids was halved (2.8±0.3% for high-fat vs. 1.4±0.1% for control, p<0.01) and was negatively correlated with intestinal permeability (r=-0.72, p=0.01, N=11). In in vitro experiments, DCA disrupted colonic barrier dose-dependently at 1–3mM concentrations, which are physiological for fat-fed-mice, but not at the lower concentrations physiological for control mice. Moreover, DCA did not increase tissue cyclooxygenase-2 content. Ursodeoxycholic acid (0.6mM) protected from barrier disruption induced by 3mM deoxycholic acid (p<0.05). High-fat feeding induces barrier dysfunction in jejunum and colon in mice. The data suggest this effect to be mediated by modifications in fecal bile acid profile. We propose altered bile acid metabolism as a novel hypothesis for the mechanism of high-fat-induced barrier dysfunction.