1,25-dihydroxyvitamin D3 (1,25(OH)(2)D-3), the active metabolite of vitamin D3 has a strong impact on the differentiation and function of immune cells. Here we analysed the influence of its precursor 25-hydroxyvitamin D3 (25(OH)D-3) on the differentiation of human CD4(+) T cells applying physiological concentrations in vitro. Our data show that 25(OH)D-3 is converted to its active form 1,25(OH)(2)D-3 by T cells, which in turn supports FOXP3, CD25 and CTLA-4 expression and inhibits IFN-gamma production. These changes were not reflected in the demethylation of the respective promoters. Furthermore, we investigated the impact of vitamin D3 metabolites under induced Treg (iTreg) polarization conditions using TGF-beta. Surprisingly, no additive effect but a decreased percentage of FOXP3 expressing cells was observed. However, the combination of 25(OH)D-3 or 1,25(OH)(2)D-3 together with TGF-beta further upregulated CD25 and CTLA-4 and significantly increased soluble CTLA-4 and IL-10 secretion whereas IFN-gamma expression of iTreg was decreased. Our data suggest that physiological levels of 25(OH)D-3 act as potent modulator of human CD4(+)T cells and autocrine or paracrine production of 1,25(OH)(2)D-3 by T cells might be crucial for the local regulation of an adaptive immune response. However, since no epigenetic changes are detected by 25(OH)D-3 a rather transient phenotype is induced.