We have previously reported that 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) can selectively suppress key functions of interferon-gamma (IFN-) activated macrophages. To further explore this mechanism for its relevance in vivo, we investigated an infection model that crucially depends on the function of IFN- activated macrophages, the infection with the intracellular protozoan Leishmania major. 1,25(OH)₂D₃ treatment of L. major infected macrophages demonstrated a vitamin D receptor (Vdr) dependent inhibition of macrophage killing activity. Further analysis showed that this was a result of decreased production of nitric oxide by 1,25(OH)₂D₃-treated macrophages due to Vdr-dependent up-regulation of arginase 1 expression, which overrides NO production by Nos2. When analyzing the course of infection in vivo, we found that Vdr-knockout (Vdr-KO) mice were more resistant to L. major infection than their wild-type littermates. This result is in agreement with an inhibitory influence of 1,25(OH)₂D₃ on the macrophage mediated host defense. Further investigation showed that Vdr-KO mice developed an unaltered T helper cell type 1 (Th1) response on infection as indicated by normal production of IFN- by CD4⁺ and CD8⁺ T cells. Therefore, we propose that the absence of 1,25(OH)₂D₃-mediated inhibition of macrophage microbicidal activity in Vdr-KO mice results in increased resistance to Leishmania infection.