Mycobacterium tuberculosis is a facultative intracellular pathogen capable of producing both progressive disease and latent infection. Latent infection is clinically asymptomatic and is manifested only by a positive tuberculin test or a chest radiograph that shows scars or calcified nodules indicative of resolved primary tuberculosis infection. In this study, we used a well‐characterized model of latent tuberculosis infection in B6D2F1 mice to compare the production of β‐defensin‐3 by infected bronchial epithelial cells and macrophages. We demonstrated by immunolectronmicroscopy that M. tuberculosis can actually infect epithelial cells and induce significant higher production of β‐defensin‐3 associated to mycobacteria than infected macrophages. These results demonstrate that lung epithelium harbour mycobacteria during experimental chronic infection; being a possible reservoir of latent mycobacteria in vivo, β‐defensins might participate in bacilli killing or dormancy induction.