Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α are the master transcription factors that regulate cellular response to hypoxia. Conflicting role of HIF-1α in regulatory T cells (Tregs) development and activity have been reported, with either that HIF-1α is obligatory for the functional Treg development or that HIF-1α is detrimental for Foxp3 stability. In this study, we re-examined the contribution of HIF-1α and HIF-2α to Tregs function using mice with T cell-specific (Cd4-Cre) knockout of HIF-1α or HIF-2α. The development of thymus-derived Tregs (tTregs) was comparable between wild-type, Hif1a-/- and Hif2a-/- mice. The expression of CTLA-4, GITR, LAG3 and FR4, as well as the production of IL-10 and TGF-β, was also similar among wild-type, Hif1a-/- and Hif2a-/- Tregs. However, we found that HIF-1α-deficient CD4+CD25+ tTregs were defective in suppressing effector T cell proliferation in vitro. HIF-1α-deficiency also impaired the differentiation of inducible regulatory T cell (iTreg) in vitro when TGF-β was not supplemented at the optimal concentration. Despite those in vitro defects of Hif1a-/- Tregs, adoptive transfer of HIF-1α-deficient tTregs into Rag1-/- mice effectively suppressed effector T cell-induced colitis. The expression of Foxp3 in Hif1a-/- tTregs was indistinguishable from WT tTregs upon recovered from the transferred mice, suggesting that the stability of Hif1a-/- Treg was normal in vivo. For HIF-2α, the suppressive function of HIF-2α-knockout tTregs was similar to WT tTregs both in vitro and in vivo. Together, our results may help resolve some discrepancies in the role of HIF-1α in Tregs, and suggest that HIF-1α or HIF-2α is not essential for the development and physiological function of Tregs.