Tight junctions create a paracellular permeability barrier, which is breached when non-steroidal anti-inflammatory drugs cause gastrointestinal injury, including increased gastrointestinal permeability. However, the mechanism by which aspirin affects the function of gastric epithelial tight junctions is unknown. Thus, we examined the effect of aspirin on gastric mucosal barrier properties and tight junction organization using MKN28, a human gastric epithelial cell line that expresses claudins-3, -4, -7, ZO-1 and occludin, but not claudin-2 or -5, as determined by immunoblotting and immunofluorescent staining. Aspirin (5 mM) treatment of MKN28 gastric epithelial monolayers significantly decreased trans-epithelial electrical resistance, and increased dextran permeability. Both aspirin-mediated permeability and phosphorylation of p38 MAP kinase were significantly attenuated by SB203580 (a p38 MAPK inhibitor), but not by U0126 (MAPK kinase (MEK) 1 inhibitor) or SP600125 (c-Jun N-terminal kinase (JNK) inhibitor). Aspirin significantly decreased the quantity of claudin-7 protein produced by the MKN28 cells, but not the quantity of claudin-3, -4, ZO-1, or occludin. The aspirin-induced decrease in claudin-7 protein was completely abolished by SB203580 pretreatment. These results demonstrate for the first time that claudin-7 protein is important in the aspirin-induced gastric barrier loss, and that p38 MAPK activity mediates this epithelial barrier dysfunction.