Mycothiol is the major low-molecular-mass thiol in M. tuberculosis, which plays an important role in maintaining redox homeostasis. Mycothiol acetyltransferase (MshD) finishes the synthesis process by transferring the acetyl group of Acetyl-CoA to the mycothiol precursor. In order to explore the role of MshD in M. tuberculosis, we constructed a MshD knockout strain with a phage vector-based homologous recombination technology. Compared with the wild strain H37Ra, the knockout had smaller colonies, slower growth rate, decreased biofilm, and was more sensitive to chemical challengers by 5 mmol/L H₂O₂, 0.05% SDS, 50 ℃ heat shock and hypoxic conditions. The above results indicated that the mshD gene played an important role in M. tuberculosis. This study laid the foundation for further revealing the function and mechanism of the gene.