Tumor microenvironment (TME) promotes immune suppression through recruiting and expanding suppressive immune cells such as regulatory T cells (Tregs) to facilitate cancer progression. In this study, we identify a novel CD39⁺ γδTreg in human colorectal cancer (CRC). CD39⁺ γδTregs are the predominant regulatory T cells and have more potent immunosuppressive activity than CD4⁺ or CD8⁺ Tregs via the adenosine-mediated pathway but independent of TGF-β or IL-10. They also secrete cytokines including IL-17A and GM-CSF, which may chemoattract myeloid-derived suppressive cells (MDSCs), thus establishing an immunosuppressive network. We further demonstrate that tumor-derived TGF-β1 induces CD39⁺ γδT cells from paired normal colon tissues to produce more adenosine and become potent immunosuppressive T cells. Moreover, CD39⁺ γδTreg infiltration is positively correlated with TNM stage and other unfavorable clinicopathological features, implicating that CD39⁺ γδTregs are one of the key players in establishment of immunosuppressive TME in human CRC that may be critical for tumor immunotherapy.