Pseudomonas aeruginosa is a major opportunistic pathogen. Host defense mechanisms involved in P. aeruginosa lung infection remains incompletely defined. Interferon regulatory factor 3 (IRF3) is a transcription factor and is primarily associated with host defense against viral infections. A role of IRF3 in P. aeruginosa infection has not been reported previously. Here we showed that IRF3 deficiency led to impaired clearance of P. aeruginosa from the lung in mice. P. aeruginosa infection induced IRF3 nucleus translocation, activation of ISRE and production of IFNβ, suggesting that P. aeruginosa induces the IRF3-ISRE-IFN pathway activation. In vitro, macrophages from IRF3 deficient mice showed complete inhibition on the production of CCL5 (RANTES) and CCL10 (IP-10), partial inhibition of CXCL1 (KC) and TNF and no effect on CXCL2 (MIP-2) in response to P. aeruginosa stimulation. In vivo, IRF3 deficient mice showed complete inhibition of CCL5 production and partial or no effects on other cytokine and chemokine production in the bronchoalveolar lavage fluids and lung tissues. Profiling of immune cells in the airways revealed that recruitment of neutrophils and macrophages into the airspace was reduced, while B cell, T cell, NK cells and NKT cells infiltrations were unaffected in IRF3 deficient mice in response to P. aeruginosa lung infection. These data suggest that IRF3 regulates a distinct profile of cytokines and chemokines and selectively modulate neutrophil and macrophage recruitment during P. aeruginosa infection. Thus, IRF3 is an integral component in the host defense against P. aeruginosa lung infection.