RORγt+Foxp3+ regulatory T (Treg) cells, known as T regulatory 17 cells (Tr17 cells), are a novel subset of Treg cells, which have the potential to regulate the development of experimental autoimmune encephalomyelitis (EAE) thorough a specific repression of T helper 17 (Th17) cell mediated inflammation. However, the function of Tr17 cells the development of other autoimmune diseases such as autoimmune arthritis remains unclear. Collagen induced arthritis (CIA) was found to be prolonged in Foxp3creRORγtfl/fl mice, in which Tr17 cells were deleted, compared with Foxp3wtRORγtfl/fl mice. Tr17 cells were significantly increased in ankle joints compared with draining lymph nodes after the onset of arthritis. CC chemokine receptor 6 (CCR6) was up-regulated on Tr17 cells compared to RORγt negative Treg cells. CD25, cytotoxic T-lymphocyte antigen 4 (CTLA-4), glucocorticoid-induced TNF-receptor (GITR), and inducible T-cell co-stimulator (ICOS) expression was also up-regulated on Tr17 cells compared to RORγt negative Treg cells. IL-10-producing cells and Blimp-1+ cells were increased in Tr17 cells compared to RORγt-Treg cells. Tr17-enriched Treg cells significantly suppressed proliferation of conventional T cells compared with CCR6-Treg cells. Tr17 cells increased during the clinical course of CIA and accumulated in inflamed joints. These cells expressed CD25, CTLA4, GITR, and ICOS molecules and up-regulated Blimp-1 and over-produced IL-10. Moreover, CCR6+ Treg cells significantly suppressed cell proliferation. Taken together, it appears that Tr17 cells play a crucial role in the regulation of autoimmune arthritis.