Mycobacterium tuberculosis, causing tuberculosis, is the pathogen that invades immune cells, especially macrophages, and evade from the host immune response. Recent studies have reported that M. tuberculosis also invade alveolar epithelial cells as well as alveolar macrophages. However, the role of alveolar epithelial cells in the host defense against M. tuberculosis remains unknown. In this study, we demonstrate that secretory leukocyte protease inhibitor (SLPI) and lipocalin 2 are secreted into the alveolar space by alveolar macrophages and epithelial cells during the early phase of respiratory mycobacterial infection. SLPI kills mycobacteria by enhancing the membrane permeability, and lipocalin 2 is internalized into the alveolar epithelial cells and inhibits intracellular mycobacterial growth by blocking iron uptake. Taken together, these findings highlight a pivotal role for alveolar epithelial cells during mycobacterial infection.