1,25-Dihydroxyvitamin D3[1,25(OH)2D3] is the biologically active form of vitamin D and is immunoregulatory. 1,25(OH)2D3binds the vitamin D receptor complex present in many immune populations and can illicit transcriptional responses that vary among different immune subsets. The effects of 1,25(OH)2D3on mature and developing human NK cells are not well characterized. In the present study, we examined the influence of 1,25(OH)2D3using an established NK cell differentiation system. Briefly, umbilical cord blood CD34+ cells were isolated and cultured in conditions optimal for NK cell differentiation, and varying concentrations of 1,25(OH)2D3were administered. At physiological concentrations (10 nM), 1,25(OH)2D3impaired NK cell development. Moreover, the NK cells that did develop under the influence of 1,25(OH)2D3showed a significant reduction in function (cytotoxicity and cytokine production). Conversely, 1,25(OH)2D3strongly induced hematopoietic stem cells to differentiate along a myeloid pathway, giving rise to CD14+ cells. Mechanistically, 1,25(OH)2D3drives hematopoietic progenitor cells to rapidly upregulate monocyte genes (i.e., C/EBP-A and CD14). There were no effects of 1,25(OH)2D3on mature NK cytotoxicity or cytokine production. Collectively, these studies provide novel data showing the negative regulatory effect of 1,25(OH)2D3on NK cell development.