Abstract: Mycothiol (MSH or AcCys-GlcN-Ins) is an unusual thiol compound found in the Actinobacteria including Actinomycetes and Mycobacterium. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against Mycobacterium tuberculosis. In this review, it is focused on MSH-dependent enzymes that utilize MSH for their activity either as a cofactor or as a substrate indicate that MSH plays a key role in protecting cells against a variety of challenges and suggest that enzymes of MSH metabolism could be possible targets for development of new drugs and vaccines directed against Mycobacterium tuberculosis and other infectious Actinomycetes.