Regulatory T-cells (Tregs) play a crucial role in maintaining immunological balance and regulating the body’s response to tumors in lymphoma patients. However, research on the role of Tregs in the biology and prognosis of splenic marginal zone lymphoma (SMZL) is lacking. CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), CyTOF (mass cytometry), and flow cytometry were used to investigate the phenotype, transcriptomic profile, and clinical significance of intratumoral Tregs and their subsets in biopsy specimens from 24 patients with SMZL and 12 reactive spleens (rSP) from individuals without lymphoma. In vitro functional experiments verified the biological properties and cell signaling pathways of intratumoral Treg subsets. Researchers discovered that Tregs are more prevalent in the spleens of SMZL patients compared to those of rSP patients and that Tregs from both SMZL and rSP patients can be subdivided into CD161+Treg and CD26+Treg populations. Although CD161+Tregs are elevated in SMZL, their immunological regulation is abnormal. Differential correlations with patient outcomes were discovered between CD161+Tregs and CD26+Tregs, suggesting that these subsets of Tregs had distinct gene expression and behavioural profiles. Patients with SMZL have an improved prognosis when their CD161+Tregs count is high, but a worse prognosis when their CD26+Tregs count is high. Not only does STAT5 inhibition counteract the effects of IL2 activation on CD26+Treg induction, but IL2 activation itself leads to the induction of CD26+Tregs. A treatment potential in SMZL may lie in the IL2/STAT5-mediated increase of CD26+Tregs, which contributes to the disease’s poor clinical prognosis.