The ability to ectopically control gene expression is a fundamental tool for the study of bacterial physiology and pathogenesis. While many efficient inducible expression systems are available for Gram-negative bacteria, few are useful in phylogenetically distant organisms, such as mycobacteria. We have adapted a highly-inducible regulon of Rhodococcus rhodochrous to artificially regulate gene expression in both rapidly-growing environmental mycobacteria and slow-growing pathogens, such as Mycobacterium tuberculosis. We demonstrate that this artificial regulatory circuit behaves as a bistable switch, which can be manipulated regardless of growth phase in vitro, and during intracellular growth in macrophages. High-level overexpression is also possible, facilitating biochemical and structural studies of mycobacterial proteins produced in their native host.