Serine acetyltransferase (CysE) belongs to the hexapeptide acetyltransferase family and is involved in the biosynthesis of L-cysteine in microorganisms. Mycobacterium tuberculosis CysE is regarded as a potential target for anti-tuberculosis (TB) drugs; however, the structure and active sites of M. tuberculosis CysE remain unknown. The present study aimed to predict the secondary structure and to construct a 3D model for M. tuberculosis CysE using bioinformatics analysis. To determine the essential amino acids that are associated with CysE enzymatic activity, amino acid sequences from several microorganisms were compared, and a consensus sequence was identified. Subsequently, site-directed mutagenesis was used to generate mutant M. tuberculosis CysE proteins. Enzyme assays demonstrated that D67A, H82A and H117A mutants abolished similar to 75% activity of M. tuberculosis CysE. Prediction of the protein structure and identification of the active amino acids for M. tuberculosis CysE is essential for designing inhibitors, which may aid the discovery of effective anti-TB drugs.