Pseudomonas aeruginosa is a Gram-negative pathogen that can lead to severe infection associated with lung injury and high mortality. The interleukin (IL)-36 cytokines (IL-36α, IL-36β and IL-36γ) are newly described IL-1 like family cytokines that promote inflammatory response via binding to the IL-36 receptor (IL-36R). Here we investigated the functional role of IL-36 cytokines in the modulating of innate immune response against P. aeruginosa pulmonary infection. The intratracheal administration of flagellated cytotoxic P. aeruginosa (ATCC 19660) upregulated IL-36α and IL-36γ, but not IL-36β, in the lungs. IL-36α and IL-36γ were expressed in pulmonary macrophages (PMs) and alveolar epithelial cells in response to P. aeruginosa in vitro. Mortality after bacterial challenge in IL-36 receptor deficient (IL-36R-/-) mice and IL-36γ deficient (IL-36γ-/-) mice, but not IL-36α deficient mice, was significantly lower than that of wild type mice. Decreased mortality in IL-36R-/-mice and IL-36γ-/-mice was associated with reduction in bacterial burden in the alveolar space, bacterial dissemination, production of inflammatory cytokines and lung injury, without changes in lung leukocyte influx. Interestingly, IL-36γ enhanced the production of prostaglandin E2 (PGE2) during P. aeruginosa infection in vivo and in vitro. Treatment of PMs with recombinant IL-36γ resulted in impaired bacterial killing via PGE2 and its receptor; EP2. P. aeruginosa infected EP2 deficient mice or WT mice treated with a COX-2-specific inhibitor showed decreased bacterial burden and dissemination, but no change in lung injury. Finally, we observed an increase in IL-36γ, but not IL-36α, in the airspace and plasma of patients with P. aeruginosa-induced acute respiratory distress syndrome. Thus, IL-36γ and its receptor signal not only impaired bacterial clearance in a possible PGE2 dependent fashion but also mediated lung injury during P. aeruginosa infection.