While Mdm2 is an important negative regulator of the p53 tumor suppressor, it also possesses p53-independent functions in cellular differentiation processes. Mdm2 expression is alternatively regulated by two P1 and P2 promoters. In this study we show that the P2-intiated transcription of Mdm2 gene is activated by 1,25-dihydroxy vitamin D3 in MC3T3 cells. By using P1 and P2-specific reporters, we demonstrate that only the P2-promoter responds to vitamin D treatment. We have further identified a potential vitamin D receptor responsive element proximal to the two p53 response elements within the Mdm2 P2 promoter. Using cell lines that are p53-temperature sensitive and p53-null, we show requirement of p53 for VDR-mediated up regulation of Mdm2 expression. Our results indicate that 1,25-dihydroxy vitamin D3 and its receptor have a role in the regulation of P2-initiated Mdm2 gene expression in a p53-dependent way. (C) 2012 Elsevier Inc. All rights reserved.