Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and an important pathogen in patients with chronic lung disease, such as cystic fibrosis and bronchiectasis. The contribution of Toll-like receptor 5 (TLR5) to the innate immune response to this organism is incompletely understood. We exposed wild-type and TLR5-deficient (Tlr5^−/−) mice to aerosolized P. aeruginosa at low and high inocula and assessed bacterial clearance, lung inflammation, and cytokine production 4 and 24 h after infection. Bacterial clearance was impaired in Tlr5^−/− mice after low-inoculum, but not high-inoculum, infection. Early bronchoalveolar accumulation of neutrophils was reduced in Tlr5^−/− mice after low- and high-dose infection. Cytokine responses, including markedly impaired monocyte chemoattractant protein-1 production 4 h after low- and high-inoculum challenge, were selectively altered in Tlr5^−/− mice. In contrast, there was no impairment of bacterial clearance, neutrophil recruitment, or monocyte chemoattractant protein-1 production in Tlr5^−/− mice after infection with a nonflagellated isotypic strain of P. aeruginosa. Thus TLR5-mediated recognition of flagellin is involved in activating pulmonary defenses against P. aeruginosa and contributes to antibacterial resistance in a manner that is partially inoculum dependent. These data are the first to demonstrate a unique role for TLR5 in the innate immune response to P. aeruginosa lung infection.