The active form of the vitamin D , 1,25-Dihydroxyvitamin D (1,25-(OH) D ) has been shown to have major effects not only on physiological processes but also on the regulation of the immune system of vertebrates. Dendritic cells are specialised antigen presenting cells which are in charge of the initiation of T-cell dependant immune responses and as such are key regulators of responses towards pathogens. In this study we set out to evaluate the effects of 1,25-(OH) D on the phenotype of cattle monocyte-derived dendritic cells (MoDCs) and how the conditioning with this vitamin affects the function of these myeloid cells. MoDCs were generated from CD14 monocytes with bovine IL-4 and GM-CSF with or without 1,25-(OH) D supplementation for 10 days. Vitamin D conditioned MoDCs showed a reduced expression of co-stimulatory and antigen presenting molecules, as well as a reduced capability of endocytose ovalbumin. Furthermore, the capacity of MoDCs to induce proliferation in an allogeneic mixed leukocyte reaction was abolished when MoDCs were generated in presence of 1,25-(OH) D . LPS induced maturation of 1,25-(OH) D conditioned MoDCs resulted in lower secretion of IL-12 and higher IL-10 than that observed in MoDCs. The typical immunotolerant phenotype observed in cattle DCs after exposure to 1,25-(OH) D has a significant effect on the functionality of these immune cells, inhibiting the T-cell stimulatory capacity of MoDCs. This could have profound implications on how the bovine immune system deals with pathogens, particularly in diseases such as tuberculosis or paratuberculosis.