Though Runx2 and Osterix are both key transcription factors in the pathway of osteoblast differentiation, whether Runx2 positively regulates Osterix being unknown. It was showed that Runx2 induced the gene expression of Osterix both in the non-osteoblastic cell lines, either pluripotent or differentiated, and in the osteoblastic cell lines. At the same time, the results also indicated that Runx2 up-regulated the activity of the 3.2 kb human Osterix promoter. Further experiments identified a highly conserved and functional Runx2 binding site “AGTGGTT” within the promoter. Thus the results support the hypothesis that Runx2 is involved in the regulation of the Osterix gene expression. Moreover, the transient transfection and dual-luciferase assay showed Osterix up-regulated the activity of the 2.3 kb typeⅠ collagen promoter in the non-osteoblastic cells, but Runx2 did not. This difference implies that Osterix, the down stream transcription factor of Runx2 during osteoblast differentiation, is needed to stimulate the osteoblast-specific gene expression of typeⅠ collagen.