Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies.Escherichia coli(E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms.E. colidoes, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogenMycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed inE. coliproduced as soluble proteins.Mycobacterium smegmatis (M. smegmatis)is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems inM. smegmatis. The advantages of usingM. smegmatisas an expression host, its application in structural biology and some practical aspects of protein production are also discussed.M. smegmatisprovides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.