We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.