Runx2 is one of the most important transcription factors directing the osteogenesis of mesenchymal stem cells and osteoblastic functions. It is likely that the factors controlling Runx2 expression would trigger the early steps of osteoblast differentiation. By using a reporter gene assay for 4.5 kb Runx2 promoter, it was found that the first 305 bp of Runx2 promoter are active in D1 cells. Within this region, electromobility shift assays (EMSAs) delineated a 6 bp of CACATG bound specifically by the proteins from D1 cell nuclear extract. Antibody super-shift and DNA-coupling magnetic bead pull-down assay indicated that the protein bound to this sequence is USF2. Site-specific mutagenesis revealed that this sequence contributed to the activity of 305 bp Runx2 promoter. Thus, we suggest that USF2 might be one of the regulators for the expression of the Runx2 gene in D1 cells.