Rationale: Cell-to-cell communication via gap junctions regulates airway epithelial cell homeostasis and maintains the epithelium host defense. Quorum-sensing molecules produced by Pseudomonas aeruginosa coordinate the expression of virulence factors by this respiratory pathogen. These bacterial signals may also incidentally modulate mammalian airway epithelial cell responses to the pathogen, a process called inter-kingdom signaling. Objectives: To investigate the interactions between the P. aeruginosa N-3-oxo-dodecanoyl-L-homoserine lactone (C12) quorum-sensing molecule and human airway epithelial cell gap junctional intercellular communication (GJIC). Methods: C12 degradation and its effects on cells were monitored in various airway epithelial cell models grown under non-polarized and polarized conditions. Its concentration was further monitored in daily tracheal aspirates of colonized intubated patients. Measurements and Main Results: C12 rapidly altered epithelial integrity and decreased GJIC in non-polarized airway epithelial cells while other quorum-sensing molecules had no effect. The effects of C12 were dependent on [Ca2+]i and could be prevented by inhibitors of Src tyrosine family and Rho-associated protein kinases. In contrast, polarized airway cells grown on Transwell filters were protected from C12, except when undergoing repair after wounding. In vivo during colonization of intubated patients, C12 did not accumulate, but paralleled bacterial densities. Interestingly, in vitro C12 degradation, a reaction catalyzed by intracellular paraoxonase 2 (PON2), was impaired in non-polarized cells whereas PON2 expression was increased during epithelial polarization. Conclusions: The cytotoxicity of C12 on non-polarized epithelial cells, combined with its impaired degradation allowing its accumulation, provide an additional pathogenic mechanism for P. aeruginosa infections.