Mycothiol <i>S</i>-transferase (MST) (encoded by the <i>rv0443</i> gene) was previously identified as the enzyme responsible for the transfer of Mycothiol (MSH) to xenobiotic acceptors in <i>Mycobacterium tuberculosis</i> (<i>M.tb</i>) during xenobiotic stress. To further characterize the functionality of MST <i>in vitro</i> and the possible roles <i>in vivo</i>, X-ray crystallographic, metal-dependent enzyme kinetics, thermal denaturation studies, and antibiotic MIC determination in <i>rv0433</i> knockout strain were performed. The binding of MSH and Zn<sup>2+</sup> increases the melting temperature by 12.9 °C as a consequence of the cooperative stabilization of MST by both MSH and metal. The co-crystal structure of MST in complex with MSH and Zn<sup>2+</sup> to 1.45 Å resolution supports the specific utilization of MSH as a substrate as well as affording insights into the structural requirements of MSH binding and the metal-assisted catalytic mechanism of MST. Contrary to the well-defined role of MSH in mycobacterial xenobiotic responses and the ability of MST to bind MSH, cell-based studies with an <i>M.tb rv0443</i> knockout strain failed to provide evidence for a role of MST in processing of rifampicin or isoniazid. These studies suggest the necessity of a new direction to identify acceptors of the enzyme and better define the biological role of MST in mycobacteria.