Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-α-D-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth, and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-D. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady state and pre-steady state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady state kinetic parameters were determined to be: kcat equal to 3.15 s−1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analog, 5′-O-[N-(L-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and non-competitive inhibition versus cysteine, with an inhibition constant of ~306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid quench techniques, giving rates of ~9.4 s−1 and ~5.2 s−1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.