Protein secretion is an essential determinant of mycobacterial virulence. Mycobacterium tuberculosis has a unique cell envelope consisting of two lipid bilayers, which requires dedicated protein secretion pathways. The conserved general Sec and Tat translocation systems are responsible for protein transport across the inner membrane and are both essential. Additionally, the accessory Sec pathway specifically contributes to virulence. How transport of Sec/Tat substrates across the outer membrane is accomplished is currently an enigma. In addition to these pathways, M. tuberculosis also developed specialized secretion systems for protein transport across both membranes, the type VII or ESX secretion systems. Here, we discuss our current knowledge about the mechanisms and substrates of these different protein translocation systems and their role in mycobacterial physiology and virulence.