Runt-related transcription factor 2 (Runx2) is essential for osteogenesis. This study aimes at identification of the genomic region differentially methylated in DNA for regulation of Runx2 expression. In the proximal promoter of mouse Runx2, DNA methylation was frequent at the region further than 3 kb relative to the transcription start site, in contrast to lower methylation status of the closer locus within 2 kb from the transcription start site. At the intermediate part, we identified a novel differentially methylated region in the Runx2 promoter region (Runx2-DMR): from -2.7 to -2.2 kb relative to the start site of Runx2 transcription in mice. In this region, the DNA methylation rate correlated negatively with Runx2 expression among mouse organs as well as among primary cultures of bone marrow from different dogs. Induction of mouse and dog mesenchymal-like cells into osteoblastic differentiation decreased the methylation rate of Runx2-DMR. Thus, in this study, we identified a novel genomic region in which DNA methylation status is related to Runx2 expression and detected demethylation of Runx2-DMR during osteoblastic differentiation in mouse and dog.