Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus Rhamnosus GG-produced soluble proteins (P40 and P75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with P40 or P75 attenuated the hydrogen peroxide-induced decrease in TER and increase in inulin permeability in a time and dose-dependent manner. P40 and P75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin and -catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both P40 and P75 induced a rapid increase in the membrane translocation of PKCI and PKC. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by P40 and P75 was abrogated by Ro-32-0432, a PKC inhibitor. P40 and P75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the P40 and P75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC and MAP kinase-dependent mechanism.