Thyrotropin receptor (TSHR) antibodies and hyperthyroidism are induced by immunizing mice with adenovirus encoding the TSHR or it's A-subunit. Depleting regulatory T cells (Treg) exacerbates thyrotoxicosis in susceptible BALB/c, and induces hyperthyroidism in normally resistant C57BL/6, mice. Vitamin D plays an important role in immunity: high dietary vitamin D intake suppresses (and low intake enhances) adaptive immune responses. Vitamin D induced immunosuppression may enhance Treg. Therefore, we hypothesized that decreased vitamin D intake would mimic Treg depletion and enhance hyperthyroidism induced by A-subunit adenovirus immunization. BALB/c mice had a reduced ability versus C57BL/6 mice to generate the active metabolite of vitamin D [1,25(OD)₂D₃ ]. Vitamin D deficiency induced subtle immune changes in BALB/c (not C57BL/6) mice. Compared with mice fed regular chow, vitamin D deprived BALB/c had fewer splenic B cells, decreased IFN- responses to mitogen, and lacked memory T cell responses to A-subunit protein. However, vitamin D deficiency did not alter TSHR antibody responses measured by ELISA, TSH binding inhibition or cAMP generation from TSHR-expressing cells. Unexpectedly, compared with vitamin D-sufficient mice, vitamin D-deficient BALB/c mice had lower pre-immunization thyroxine levels and developed persistent hyperthyroidism. This difference was unrelated to the immunological changes between vitamin D deficient - or sufficient- animals. Previously, we found that different chromosomes or loci confer susceptibility to TSHR antibody induction versus thyroid function. Our present studies provide evidence that an environmental factor, vitamin D, has only minor effects on induced immunity to the TSHR, but directly affects thyroid function in mice.