To study the role of invariant Natural Killer T cell (iNKT) cells in autoimmune thyroiditis, we derived two iNKT cell lines from the spleens of NOD·H2h4mice, a strain that develops spontaneous autoimmune thyroiditis exacerbated by excess dietary iodine. The two lines were CD1d-restricted and expressed CD4+, DX5+, and the Vα4Jα281 gene segment, of the T-cell receptor α locus. Upon stimulation with α-galactosyl-ceramide (α-GalCer), both lines rapidly produced IL-2, IL-4, IFN-γ, IL-10, and TNF-α. Strikingly, a similar cytokine response was also induced by thyroglobulin, one of the most abundant protein in the thyroid gland and a major autoantigen in human autoimmune thyroiditis. Transfer of the iNKT cell lines to syngeneic hosts enhanced autoimmune thyroiditis. Intraperitoneal injections of α-GalCer in iodine primed mice also induced thyroid disease. This paper reports for the first time that iNKT cells respond to thyroglobulin and enhance autoimmune thyroiditis in iodine fed NOD·H2h4mice.