Abstract: The objectives of this study were to (a) evaluate and compare the ability of ex vivo-generated induced regulatory T cells (iTregs) and freshly isolated natural Tregs (nTregs) to reverse/attenuate preexisting intestinal inflammation in a mouse model of chronic colitis and (b) quantify the Treg-targeted gene expression profiles of these two Treg populations. We found that ex vivo-generated iTregs were significantly more potent than nTregs at attenuating preexisting colitis. This superior therapeutic activity was associated with increased accumulation of iTregs within the mesenteric lymph nodes and large and significant reductions in interleukin (IL)-6 and IL-17A expression in the colons of iTreg- versus nTreg-treated mice. The enhanced immunosuppressive activity of iTregs was not because of increased expression or stability of Foxp3 as iTregs and nTregs obtained from the mesenteric lymph nodes, and colons of reconstituted mice expressed similar levels of this important transcription factor. In addition, we observed a total of 27 genes that were either upregulated or downregulated in iTregs when compared with nTregs. Although iTregs were found to be superior at reversing established disease, their message levels of IL-10 and IL-35 and surface expression of the gut-homing molecules CCR9 and α4β7 were significantly reduced when compared with nTregs. Taken together, our data demonstrate that ex vivo-generated iTregs are significantly more potent than nTregs at attenuating preexisting gut inflammation despite reduced expression of classical regulatory cytokines and gut-homing molecules. Our data suggest that the immunosuppressive activity of iTregs may be because of their ability to directly or indirectly decrease expression of IL-6 and IL-17A within the inflamed bowel.