The expression of BTB-ZF transcription factors such as ThPOK in CD4+ T cells, Bcl6 in T follicular helper cells, and PLZF in natural killer T cells defines the fundamental nature and characteristics of these cells. Screening for lineage-defining BTB-ZF genes led to the discovery of a subset of T cells that expressed Zbtb20. About half of Zbtb20+ T cells expressed FoxP3, the lineage-defining transcription factor for regulatory T cells (Tregs). Zbtb20+ Tregs were phenotypically and genetically distinct from the larger conventional Treg population. Zbtb20+ Tregs constitutively expressed mRNA for interleukin-10 and produced high levels of the cytokine upon primary activation. Zbtb20+ Tregs were enriched in the intestine and specifically expanded when inflammation was induced by the use of dextran sodium sulfate. Conditional deletion of Zbtb20 in T cells resulted in a loss of intestinal epithelial barrier integrity. Consequently, knockout (KO) mice were acutely sensitive to colitis and often died because of the disease. Adoptive transfer of Zbtb20+ Tregs protected the Zbtb20 conditional KO mice from severe colitis and death, whereas non-Zbtb20 Tregs did not. Zbtb20 was detected in CD24hi double-positive and CD62Llo CD4 single-positive thymocytes, suggesting that expression of the transcription factor and the phenotype of these cells were induced during thymic development. However, Zbtb20 expression was not induced in "conventional" Tregs by activation in vitro or in vivo. Thus, Zbtb20 expression identified and controlled the function of a distinct subset of Tregs that are involved in intestinal homeostasis.