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<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) is an important and harmful intracellular pathogen that is responsible for the cause of tuberculosis (TB). <i>Mtb</i> capsular polysaccharides can misdirect the host’s immune response pathways, resulting in additional challenges in TB treatment. These capsule polysaccharides are biosynthesized by stealth proteins, including CpsY. The structure and functional mechanism of <i>Mtb</i> CpsY are not completely delineated. Here, we reported the crystal structure of CpsY^201−520 at 1.64 Å. CpsY^201−520 comprises three β-sheets with five α-helices on one side and three on the other. Four conserved regions (CR1–CR4) are located near and at the base of its catalytic cavity, and three spacer segments (S1–S3) surround the catalytic cavity. Site-directed mutagenesis demonstrated the strict conservation of R419 at CR3 and S1–S3 in regulating the phosphotransferase activity of CpsY^201−520. In addition, deletion of S2 or S3 (∆S2 or ∆S3) dramatically increased the activity compared to the wild-type (WT) CpsY^201−520. Results from molecular dynamics (MD) simulations showed that S2 and S3 are highly flexible. Our study provides new insights for the development of new vaccines and targeted immunotherapy against <i>Mtb.</i>