NOD.H-2h4 mice given NaI in their drinking water develop iodine-accelerated spontaneous autoimmune thyroiditis (ISAT) with chronic inflammation of the thyroid by T and B cells and production of anti-mouse thyroglobulin (MTg) autoantibody. CD28-/-NOD.H-2h4 mice, which have reduced numbers of CD4+Foxp3+regulatory T cells (Tregs), were developed to examine the role of Tregs in ISAT development. CD28-/-NOD.H2-h4 mice develop more severe ISAT than do wild-type (WT) mice, with collagen deposition (fibrosis) and low serum T4. CD28-/-mice have increased expression of proinflammatory cytokines IFN-g and IL-6, consistent with increased mononuclear cell infiltration and tissue destruction in thyroids. Importantly, transferring purified CD4+Foxp3+Tregs from WT mice reduces ISAT severity in CD28-/-mice without increasing the total number of Tregs, suggesting that endogenous Tregs in CD28-/-mice are functionally ineffective. Endogenous CD28-/-Tregs have reduced surface expression of CD27, TNFR2 p75, and glucocorticoid-induced TNFR-related protein compared with transferred CD28+/+ Tregs. Although anti-MTg autoantibody levels generally correlate with ISAT severity scores in WT mice, CD28-/-mice have lower anti-MTg autoantibody responses than do WT mice. The percentages of follicular B cells are decreased and those of marginal zone B cells are increased in spleens of CD28-/-mice, and they have fewer thyroid-infiltrating B cells than do WT mice. This suggests that CD28 deficiency has direct and indirect effects on the B cell compartment. B cell-deficient (B-/-) NOD.H-2h4 mice are resistant to ISAT, but CD28 -/-B-/-mice develop ISAT comparable to WT mice and have reduced numbers of Tregs compared with WT B-/-mice. The Journal of Immunology, 2013, 191:4940-4949.