Objective: Changes in epithelial tight junction protein expression and apoptosis increase epithelial permeability in inflammatory bowel diseases. The effect of the probiotic mixture VSL#3 on the epithelial barrier was studied in Dextran-Sodium-Sulphate (DSS)-induced colitis in mice. Material and Methods: Acute colitis was induced in balb/c mice (3.5 % DSS for 7 days). Mice were treated with either 15 mg VSL#3 or placebo via gastric tube once daily during induction of colitis. Inflammation was assessed by clinical and histological scores. Colonic permeability to Evans blue was measured in vivo. Tight junction protein expression and epithelial apoptotic ratio were studied by immunofluorescence and Western blot. Results: VSL#3 treatment reduced inflammation (histological colitis scores: healthy control 0.94 ± 0.28, DSS + placebo 14.64 ± 2.55, DSS + VSL#3 8.43 ± 1.82; p=0.011). A pronounced increase in epithelial permeability in acute colitis was completely prevented by VSL#3 therapy (healthy control 0.4 ± 0.07 (Ext./g), DSS + placebo 5.75 ± 1.67, DSS + VSL#3 0.26 ± 0.08; p=0.003). In acute colitis, decreased expression and redistribution of the tight junction proteins occludin, ZO-1, claudin-1, -3, -4, and -5 were observed, whereas VSL#3 therapy prevented these changes. VSL#3 completely prevented the increase of epithelial apoptotic ratio in acute colitis (healthy control 1.58 ± 0.01 (apoptotic cells / 1000 epithelial cells), DSS + placebo 13.33 ± 1.29, DSS + VSL#3 1.72 ± 0.1; p=0.012). Conclusions: Probiotic therapy protects the epithelial barrier in acute colitis by preventing 1) decreased tight junction protein expression, 2) increased apoptotic ratio.