Mycobacterium tuberculosis, the causative agent of tuberculosis, is among the most successful human pathogens. Throughout their complex life cycle in the host – infection, growth, transmission – mycobacteria must continually resist host defences. A major mycobacterial survival mechanism lies in the unusual and complex cell envelope. The mycobacterial cell envelope provides a robust permeability barrier that helps the bacteria tolerate the variety of host insults it encounters. Many mycobacterial virulence factors are localised in the cell envelope. Here I have dissected the function of Erp (exported repetitive protein), a cell envelope associated, critical virulence factor, that is required for survival and growth specifically in host macrophages. I find that Erp appears to anchor the cell wall to the inner membrane, thereby preserving cell envelope ultrastructure. Localisation of Erp specifically to the cell wall is required to maintain the cell envelope as an impermeable barrier. Loss of Erp increases the permeability of the cell envelope, sensitising mycobacteria to both hydrophobic antibiotics and a range of antimicrobial molecules enriched in macrophages. Erp is mycobacterium-specific, and widely conserved in both saprophytic and pathogenic mycobacteria, suggesting that it co-evolved with the mycobacterial cell envelope to maintain a permeability barrier that was essential to counter ubiquitous environmental antimicrobial factors. Erp’s function then allowed mycobacteria to evolve into intracellular pathogens.