CD1d-restricted natural killer T (NKT) cells and CD4CD25 regulatory T (Treg) cells are two thymus-derived subsets of regulatory T cells that play an important role in the maintenance of self-tolerance. Yet the functional changes of the two subsets of regulatory T cells in the development of diabetes in non-obese diabetic (NOD) mice remain unclear, and how NKT cells and CD4CD25 Treg cells cooperate functionally in the regulation of autoimmune diabetes is also uncertain. We provide evidence that in NOD mice, an animal model of human type 1 diabetes, the functions of both NKT cells and CD4CD25 Treg cells decrease in an age-dependent manner. We show that treatment with α-galactosylceramide increases the size of the CD4CD25 Treg cell compartment in NOD mice, and augments the expression of forkhead/winged helix transcription factor and the potency of CD4 CD25 Treg cells to inhibit proliferation of CD4 CD25 T cells. Our data indicate that NKT cells and CD4CD25 Treg cells might cooperate in the prevention of autoimmune diabetes in NOD mice treated with α-galactosylceramide. Induced cooperation of NKT cells and CD4CD25 Treg cells could serve as a strategy to treat human autoimmune disease, such as type 1 diabetes. © 2008 Institute of Biochemistry and Cell Biology, SIBS, CAS.