The vitamin D receptor (VDR) and its ligand 1,25-OH2-VD3 (calcitriol) play an essential role in mineral homeostasis in mammals. Interestingly, the VDR is expressed very early in adipogenesis in 3T3-L1 cells suggesting that the VDR signaling pathway may play a role in adipocyte biology and function. Indeed, it has been known for a number of years that calcitriol is a potent inhibitor of adipogenesis in this model but with no clear mechanism identified. In this study we have further defined the molecular mechanism by which the unliganded VDR and calcitriol-liganded VDR regulate adipogenesis. In the presence of calcitriol, the VDR blocks adipogenesis by down-regulating both C/EBPß mRNA expression and C/EBPß nuclear protein levels at a critical stage of differentiation. In addition, calcitriol allows for the upregulation of the recently described C/EBPß corerepressor, ETO, which would further inhibit the action of any remaining C/EBPß, whose action is required for adipogenesis. In contrast, in the absence of calcitriol, the unliganded VDR appears necessary for lipid accumulation as knockdown of the VDR using siRNA both delays and prevents this process. Taken together these data support the notion that the intracellular concentrations of calcitriol can play an important role in either promoting or inhibiting adipogenesis via the VDR and the transcriptional pathways which it targets. Further examination of this hypothesis in vivo may shed new light on the biology of adipogenesis.