Defective hydration of airway surface mucosa is associated with recurrent lung infection in cystic fibrosis (CF), a disease caused by CF transmembrane conductance regulator (CFTR) gene mutations. Whether the composition and/or presence of an airway surface liquid (ASL) is sufficient to prevent infection remains unclear. The susceptibility to infection of polarized wild type andCFTRknockdown (CFTR-KD) airway epithelial cells was determined in the presence or absence of a healthy ASL or physiological saline. CFTR-KD epithelia exhibited strong ASL volume reduction, enhanced susceptibility to infection, and reduced junctional integrity. Interestingly, the presence of an apical physiological saline alleviated disruption of the airway epithelial barrier by stimulating essential junctional protein expression. Thus, rehydrated CFTR-KD cells were protected from infection despite normally intense bacterial growth. This study indicates that an epithelial integrity gatekeeper is modulated by the presence of an apical liquid volume, irrespective of the liquid's composition and of expression of a functional CFTR.