The effects of lactobacilli on impaired intestinal barrier function and paracellular permeability were evaluated in human epithelial Caco-2 cells treated with tumor necrosis factor-alpha and in mice with colitis induced by dextran sodium sulfate (DSS). Filter-grown Caco-2 monolayers were used as the intestinal epithelial model. Among the 4 lactobacilli studied, Lactobacillus rhamnosus OLL2838 most effectively suppressed barrier impairment and increased IL-8 secretion induced by tumor necrosis factor-alpha in Caco-2 cells; however, the conditioned medium from OLL2838 did not show any effect on barrier functions. The in vivo effects of OLL2838 on intestinal epithelial barrier function and colonic inflammation were assessed in DSS-induced colitis of BALB/c mice. Oral treatment with both live and heat-killed OLL2838 suppressed weight loss and recovered colon length. Additionally, barrier function was restored by the administration of live and heat-killed OLL2838 to the DSS-treated animals, which conferred protection against the increase in mucosal permeability associated with DSS-induced colitis. This may at least partially be because of the increased expression of zonula occludens-1 (4.8-fold) and myosin light-chain kinase (3.1-fold) in intestinal epithelial cells isolated from mice of the heat-killed OLL2838 group. Therefore, L. rhamnosus OLL2838 would be useful in the treatment of gastrointestinal diseases such as inflammatory bowel disease.