1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), a key regulator of mineral metabolism, regulates the expression of several genes that are expressed in osteoblasts. In particular, in rat and human osteoblasts, 1,25-(OH)2D3 increases the expression of Osteocalcin by interacting, through a hormone-receptor complex, with a vitamin D-responsive element present in the promoter of the genes. Here we show that in mouse, 1,25-(OH)2D3 inhibits the expression of both osteocalcin genes, OG1 and OG2. This inhibition was observed in primary osteoblast cultures and in the whole animal. From sequence inspection, DNA transfection experiments, and DNA binding assays, we could not identify a functional vitamin D-responsive element in the promoter of OG2 or in the first 3.3 kilobases of the OG1 promoter. However, we show that 1,25-(OH)2D3 treatment of primary osteoblasts abolishes the binding of OSF2, an osteoblast-specific activator of transcription that binds to OSE2, a critical osteoblast-specific cis-acting element present in OG1 and OG2 promoters. Consistent with these DNA binding data, a mutation in OSE2 in the OG2 promoter abrogated the inhibitory effect of 1,25-(OH)2D3 treatment on this promoter activity. This study illustrates that 1,25-(OH)2D3 can play different roles in the expression of the same gene in various species and indicates that this regulation in mouse occurs through an indirect mechanism, 1,25-(OH)2D3 acting on a gene genetically located upstream of Osteocalcin.