TNF-α has a multifunctional role in autoimmune diseases as reflected in the variable responses of different human diseases to anti-TNF-α therapy. Recent studies have suggested that TNF-α modulates autoimmunity partially via effects on regulatory T cells (Tregs) and that these effects are mediated through the type II TNFR (TNFR2). We have investigated the requirement for TNFR2-expression on murine natural Tregs (nTregs) and induced Tregs (iTregs) in mediating suppression of colitis. Surprisingly, we find that TNFR2-expression is required for both spleen- and thymus-derived nTreg-mediated suppression, but is not required for iTreg-mediated suppression. Abnormal TNFR2(-/-) nTreg function was not associated with an in vivo decrease in accumulation, stability, or expression of markers known to be relevant in Treg function. Because iTregs are generated in the presence of TGF-β, we investigated whether activation in the presence of TGF-β could overcome the functional defect in TNFR2(-/-) nTregs. Although preactivation alone did not restore suppressive function of nTregs, preactivation in the presence of TGF-β did. These results identify potentially critical differences in activation requirements for nTregs versus iTregs. Furthermore, our findings are consistent with reports suggesting that nTregs are activated in sites of inflammation while iTregs are activated in lymph nodes. Finally, by demonstrating that nTregs require TNF-α for optimal function whereas iTregs do not, our results suggest that the enigma of variable responses of different human diseases to anti-TNF-α therapy may relate to whether nTregs or iTregs have the predominant regulatory role in a given disease.