Although reactive oxygen species have been implicated as mediators of gastrointestinal injury, their influence on the function of gastric epithelial tight junctions (TJs), which create a paracellular permeability barrier, needs to be fully investigated. H2O2 exposure to MKN28 gastric epithelial monolayers caused a significant decrease in trans-epithelial electrical resistance (TEER) and a significant increase in dextran permeability. Oxidant-mediated gastric epithelial permeability was significantly attenuated by a radical scavenger, rebamipide. H2O2 decreased the amount of claudin-3 protein but not claudin-4, -7, and JAM-A. Rebamipide significantly attenuated H2O2-induced decrease in claudin-3 protein. Small interfering RNA (siRNA) against claudin-3 treatment specifically decreased claudin-3 as seen by immunoblotting and immunofluorescent staining. Gastric TEER was significantly decreased with the treatment of siRNA against claudin-3. This is the first study to demonstrate that claudin-3 is involved in the barrier function of gastric epithelial cells and that rebamipide abolishes the H2O2-induced decrease in claudin-3 protein.