Mycobacterium　tuberculosis　(Mtb)　is　an　important　and　harmful　intracellular　pathogen　that　is　responsible　for　the　cause　of　tuberculosis　(TB).　Mtb　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　Mtb　CpsY　are　not　completely　delineated.　Here,　we　reported　the　crystal　structure　of　CpsY201−520　at　1.64　Å.　CpsY201−520　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　CpsY201−520.　In　addition,　deletion　of　S2　or　S3　(∆S2　or　∆S3)　dramatically　increased　the　activity　compared　to　the　wild-type　(WT)　CpsY201−520.　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　Mtb.　©　2023　by　the　authors.