Intact alveolar barrier function is associated with better outcomes in acute lung injury patients; however, the regulation of alveolar epithelial paracellular transport during lung injury has not been extensively investigated. This study was undertaken to determine whether changes in tight junction claudin expression affect alveolar epithelial barrier properties, and to determine the mechanisms of altered expression. In anesthetized mice exposed to ventilator-induced lung injury, claudin 4 was specifically induced among tight junction structural proteins. Real time PCR showed an 8-fold increase in claudin 4 expression in the lung injury model. To examine the role of this protein in barrier regulation, claudin 4 function was inhibited with siRNA and a blocking peptide derived from the binding domain of Clostridium perfringens enterotoxin (CPE_BD). Inhibition of claudin 4 decreased transepithelial electrical resistance, but did not alter macromolecule permeability in primary rat and human epithelial cells. In mice, CPE_BD decreased airspace fluid clearance over 33% and resulted in pulmonary edema during moderate tidal volume ventilation that did not induce edema in control peptide-treated mice. In vitro, phorbol ester induced a 9-fold increase in claudin 4 expression that was dependent on protein kinase C activation and the JNK MAPK pathway. These data establish that changes in alveolar epithelial claudin expression influence paracellular transport, alveolar fluid clearance rates, and susceptibility to pulmonary edema. We hypothesize that increased claudin 4 expression early in acute lung injury represents a mechanism to limit pulmonary edema, and that the regulation of alveolar epithelial claudin expression may be a novel target for acute lung injury therapy.