We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] inhibits adipogenesis in vitro. 1,25(OH)₂D₃ blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24 to 48 hours after the differentiation is initiated, suggesting that 1,25(OH)₂D₃ inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)₂D₃ does not block the mitotic clonal expansion or C/EBP induction; rather, 1,25(OH)₂D₃ blocks the expression of C/EBP, PPAR, SREBP-1 and other downstream adipocyte markers. The inhibition by 1,25(OH)₂D₃ is reversible, as removal of 1,25(OH)₂D₃ from the media restores the adipogenic process with only a temporal delay. Interestingly, while the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 pre-adipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 hours and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated over-expression of hVDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)₂D₃ is ameliorated by troglitazone, a specific PPAR antagonist; conversely, hVDR partially suppresses the transacting activity of PPAR, but not of C/EBP or C/EBP. Moreover, 1,25(OH)₂D₃ markedly suppresses C/EBP and PPAR mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)₂D₃ occurs at the post-clonal expansion stages and involves direct suppression of C/EBP and PPAR] up-regulation, antagonizing of PPAR activity and stabilization of the inhibitory VDR protein.