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The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX‐1–ESX‐5). While the role of the ESX‐1 and ESX‐3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX‐5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX‐5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX‐5 knockout/deletion mutants, inactivating eccA₅, eccD₅, rv1794 and esxM genes or the ppe25‐pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX‐5‐encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD₅ko mutant also showed enhanced sensitivity to detergents …