Mycobacteria have a highly special cell envelope that separates them from both Gram-positive and Gram-negative bacteria. To be able to secrete protein across this highly special cell envelope mycobacteria use type VII secretion (T7S) systems. Pathogenic mycobacteria, such as the notorious pathogen Mycobacterium tuberculosis, have up to five of these secretion systems, named ESX-1 to ESX-5. At least three of these secretion systems are essential for mycobacterial virulence and/or viability. Elucidating T7S is therefore essential to understand the success of M. tuberculosis and other pathogenic mycobacteria and could be instrumental to identify novel targets for drug- and vaccine-development. In collaboration with different (inter)national research teams we study the T7S machinery, the secreted substrates and the function of these substrates. Recently, we have been able to show the first structure of a mycobacterial T7S membrane complex obtained by single-particle analysis (Beckham et al., Nat Microbiol 2017).