Vitamin D is hypothesised as central to immune regulation and may subsequently curtail the development of autoimmunity and allergy. Vital to the immunomodulatory effects of vitamin D are regulatory T cells. Active vitamin D (1,25-dihydroxyvitamin D, 1,25(OH)2D) can increase the numbers and suppressive capacity of Foxp3+ regulatory T (TReg) cells through release of immunosuppressive cytokines like IL-10 and enhanced expression of immunoregulatory proteins CTLA-4 and Foxp3. Many of the studies of the effects of vitamin D on TReg cell activity have been performed in vitro with non-physiological concentrations of 1,25(OH)2D (nM range), and there is doubt as to whether these processes occur with physiologically-relevant 1,25(OH)2D (pM range). However, there is emerging evidence that circulating TReg cell numbers and activity are positively correlated with serum 25-hydroxyvitamin D (25(OH)D) levels in both healthy people and individuals with autoimmune and allergic disease. In addition, vitamin D may also determine the homing capacity of TReg cells, with the expression of tissue-homing molecules particularly for the skin determined by circulating 25(OH)D. We used a physiologically-relevant murine model of developmental vitamin D-deficiency to determine whether there are any tissue-specific effects of circulating 25(OH)D on TReg cell number and function. Long-term vitamin D deficiency impaired the suppressive capacity of TReg cells at multiple sites, specifically reduced the number of TReg cells in skin-draining lymphatic tissue, but had the opposite effect in the airway-draining lymph nodes. Vitamin D may thus promote TReg cell migration/development for the specific control of autoimmune and allergic immune responses that are initiated in the skin.