Autoimmune diseases are characterized by an imbalance between regulatory T cells and effector T-cell subsets, such as Th1 and Th17 cells. Studies have confirmed that natural CD4+ Foxp3+ Tregs were unstable and dysfunctional in the presence of pro-inflammatory cytokines. In the current study, human CD39hi Tregs and CD39low Tregs were sorted from Tregs in vitro after 7 days of expansion. The functions of both Treg subsets were investigated under inflammatory conditions in vitro and in vivo. In the presence of IL-1β and IL-6, cultured CD4+ CD39hi Tregs maintained stable forkhead box protein 3 expression, whereas CD4+ CD39low Tregs lost Foxp3 expression and trans-differentiated into Th1 or Th17 cells. Decreased IL-1βR and IL-6R expression on the CD39hi Tregs was the primary mechanism responsible for Treg stability. In addition, reduced activation of downstream molecules, such as STAT1 and STAT3, through the modulation of CpG demethylation played an important role. Finally, human CD4+ CD39hi Tregs but not CD4+ CD39low Tregs protected against xenograft versus host disease in model mice. These results strongly implied the physiological importance of CD39 expression and suggested that manipulation of CD39hi Tregs might represent a novel strategy for the treatment of autoimmune diseases.