Epithelial cells have the ability to regulate paracellular permeability dynamically in response to extracellular stimuli. With every respiratory effort, airway epithelial cells are exposed to both physiologic as well as pathologic stimuli, and regulation of the epithelial barrier in response to these stimuli is crucial to respiratory function. We report that increased membrane septin-2 localization mediates decreases in paracellular permeability by altering cortical actin arrangement in human airway epithelial cells. This phenomenon occurs in response to both physiologic levels of shear stress and a pathologic stimulus, particular matter exposure. The resulting changes in barrier function in response to septin-2 redistribution have a significant impact on the ability of the apical ligand, epidermal growth factor, to interact with its receptor, epidermal growth factor receptor, which is segregated to the basolateral side in airway epithelial cells. This suggests that the dynamic regulation of the epithelial barrier function is essential in regulating signaling responses to extracellular stimuli. These findings indicate that septin-2 plays a fundamental role in regulating barrier function by altering cortical actin expression.