In order to validate and estimate the capability of BMP9 to induce osteogenic differentiation of multipotent stem cells, three multipotent stem cells (C3H10, MEFs and BMSC) were used as target cells, and BMP9 was introduced into these cells by using recombinant adenoviruses assay, the effect of BMP9 on osteogenic differentiation of multipotent stem cells was demonstrated by using luciferase reporter assay, alkaline phosphatase(ALP) quantitative assay, calcium deposition assay, real time PCR, animal experiment and histological staining assay. The results demonstrated that BMP9 can induce ALP expression of C3H10, MEFs and BMSC by a dose dependent manner. BMP9 can also stimulate calcium deposition of C3H10 and MEFs in vitro, the osteogenic markers (ALP, Runx2, osteopontin, osteocalcin) were increased after stimulated by BMP9. BMP9 can activate canonical TGFβ-Smad pathway, and promote the expression of osteogenic master gene Runx2. The animal experiment and histological staining assay show that BMP9 can induce ectopic bone formation in naked mice. To sum up, BMP9 is a more powerful cell factor to induce osteogenic differentiation of multipotent stem cells.