Enhanced iodide ingestion is known to accelerate the incidence and severity of spontaneous autoimmune thyroiditis [iodide-accelerated spontaneous autoimmune thyroiditis (ISAT)] in NOD.H2(h4) mice. CD4+ cells are required for the development and maintenance of ISAT, but their target epitopes remain unknown. In this study, we show that the previously identified thyroglobulin (Tg) T cell epitope p2549-2560 containing thyroxine at position 2553 (T4p2553) induces thyroiditis as well as strong specific T and B cell responses in NOD.H2(h4) mice. In ISAT, activated CD4+ T cells specific for T4p2553 are detected before the disease onset in thyroid-draining cervical lymph nodes only in mice placed on an iodide-rich diet and not in age-matched controls. In addition, selective enrichment of CD4+ IFN-γ+ T4p2553-specific cells is observed among cervical lymph node cells and intrathyroidal lymphocytes. T4p2553 was equally detectable on dendritic cells obtained ex vivo from cervical lymph node cells of NaI-fed or control mice, suggesting that the iodide-rich diet contributes to the activation of autoreactive cells rather than the generation of the autoantigenic epitope. Furthermore, spontaneous T4p2553-specific IgG are not detectable within the strong Tg-specific autoantibody response. To our knowledge, these data identify for the first time a Tg T cell epitope as a spontaneous target in ISAT.