Ulcerative colitis (UC) is characterized by increased epithelial cell death and subsequent breakdown of the intestinal epithelial barrier, which perpetuates chronic intestinal inflammation. Since fecal bile acid dysmetabolism is associated with UC and tauroursodeoxycholic acid (TUDCA) has been shown to improve murine colitis, we evaluated the effect of TUDCA on intestinal epithelial cell death in a mouse model of UC-like barrier dysfunction elicited by dextran sulfate sodium (DSS). We identified the prevention of colonic caspase-3 induction, a key proapoptotic marker which was also over-activated in UC, as the earliest event resulting in a clear clinical benefit. Whereas vehicle-treated mice showed a cumulative mortality of 40%, all TUDCA-treated mice survived the DSS experiment during a 14-day follow-up period. In line with a barrier protective effect, TUDCA decreased bacterial translocation to the spleen and stimulated mucin production. Similarly, TUDCA inhibited lipopolysaccharide-induced intestinal permeability and associated enterocyte apoptosis. The anti-apoptotic effect was confirmed in vitro by a dose-dependent inhibition of both receptor-dependent (using tumor necrosis factor and Fas ligand) and receptor-independent (staurosporine) caspase-3 induction in HT29 colonic epithelial cells. These data imply that caspase-3 activation is an early marker of colitis that is prevented by TUDCA treatment. These data, together with the previously reported beneficial effect in colitis, suggest that TUDCA could be an add-on strategy to current immunosuppressive treatment of UC patients.