The exact effect of probiotics on epithelial barrier function is not well understood. The aims of this study were to evaluate cytokine-induced epithelial barrier dysfunction in intestinal epithelial cells (IECs) and to study the role of probiotics in the prevention of epithelial barrier dysfunction. Caco-2 cells grown on transwell chambers were stimulated with tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma basolaterally. Probiotic, Lactobacillus casei, was added 1 h before cytokine stimulation. MAPK inhibitors were added 15 min before L. casei stimulation. The electrical resistance and paracellular permeability of Caco-2 monolayers were measured. Distribution of zonula occludens (ZO)-1 protein was assessed by immunofluorescence, and Western blot analyses for ZO-1, p-Akt, and toll-like receptor (TLR) 2 were performed. Both TNF-alpha and IFN-gamma stimulation on Caco-2 cells decreased transepithelial resistance (TER), increased epithelial permeability, and decreased ZO-1 expression of Caco-2 cells. In contrast, pretreatment of L. casei reversed the cytokine-induced dysfunction of TER, epithelial permeability, and ZO-1 expression. Reversal of cytokine-induced dysfunction of TER and intestinal permeability by L. casei was abrogated with MAPK inhibitor treatment. Lactobacillus casei stimulation on Caco-2 cells increased TLR2 and p-Akt expression. Probiotic, L. casei, prevents cytokine-induced epithelial barrier dysfunctions in IECs.