Rationale: Effective protective immunity against respiratory infections with intracellular pathogens requires pathogen-specific cytotoxic T cells (CTL) in the lung. However, vaccines that induce pathogen-specific CTL in the lung are poorly explored. Dendritic cells (DC) have increasingly been exploited as vaccines against infections. However, few studies have investigated the ability of mucosal DC vaccines to elicit protective CTL responses in the lung. Objectives: To develop an efficacious mucosal DC vaccine to generate protective CTL against respiratory infections with intracellular pathogens. Methods: Bone marrow-derived DC (BM-DC) pulsed with a single immunodominant CTL epitope, listeriolysin O (LLO) 91-99, of Listeria monocytogenes (LM) were intratracheally administered into mice. The frequency and function of epitope-specific CTL in mediastinal lymph nodes (MLN) and spleen were assessed for their ability to protect against LM infection. Measurements and Main Results: Following intratracheal administration, lipopolysaccharide (LPS)-treated LLO 91-99-loaded BM-DC (LPS-LLO DC) more frequently migrated to MLN than LPS-untreated LLO 91-99-loaded BM-DC (LLO DC). Using tetrameric H2-K^d/LLO 91-99 peptide complex, specific CD8⁺ T cells were found in MLN as well as the spleen in LPS-LLO DC-immunized mice, but not in LLO-DC-immunized mice. Both MLN and spleen cells obtained from LPS-LLO DC-immunized mice produced large amounts of interferon- in response to LLO 91-99 with high epitope-specific CTL activities. Vaccination with LPS-LLO DC, but not LLO DC, protected mice against lethal respiratory infection with LM. Conclusions: These data suggest that mucosal vaccination with LPS-treated immunodominant CTL epitope-loaded DC is a promising strategy for generating protective CTL against respiratory infections with intracellular pathogens.