CYP27A1 catalyses hydroxylations in the biosynthesis of bile acids and the bioactivation of vitamin D3. We investigated the expression of CYP27A1 in human monocytes, monocyte-derived macrophages, and dendritic cells on mRNA and protein levels as well as its enzymatic activity in comparison with the expression of CYP27B1 and CYP24A1. Macrophages showed a strong expression of CYP27A1, whereas monocytes and dendritic cells expressed low levels of CYP27A1 mRNA. Immunohistochemistry revealed CYP27A1 and CYP27B1 protein expression in macrophages. Accordingly, macrophages converted vitamin D3 into the active metabolite 1,25(OH)2D3. Dendritic cells also metabolized vitamin D3 although to a lesser extent. This could be due to the high expression of CYP24A1, the enzyme that degrades 25(OH)D3 and 1,25(OH)2D3. Our results show that macrophages and dendritic cells are capable to perform both hydroxylation steps of the vitamin D3 metabolism suggesting a possible role of local 1,25(OH)2D3 synthesis by myeloid cells in the skin and gut.