Mycobacterium smegmatis (M. smegmatis) is a Gram­positive saprophytic bacterium, with the characteristics of fast­growing, non­pathogenicity as well as the highly homologous genes and similar cellular structure with Mycobacterium tuberculosis (M. tuberculosis). M. smegmatis is an ideal model for mycobacterial infection and other related immune and infectious diseases. The applications of M. smegmatis in anti­tuberculosis infection, immunoregulation and control of non­tuberculosis infection are reviewed in the present paper.