We have found that the previously uncharacterized bone morphogenetic protein-9 (BMP9) is one of the most osteogenic factors. However, it is unclear if BMP9 cross-talks with $TGF{eta}1$ during osteogenic differentiation. Using the recombinant BMP9 adenovirus, we find that low concentration of rh$TGF{eta}1$ synergistically induces alkaline phosphatase activity in BMP9-transduced C3H10T1/2 cells and produces more pronounced matrix mineralization. However, higher concentrations of $TGF{eta}1$ inhibit BMP9-induced osteogenic activity. Real-time PCR and Western blotting indicate that BMP9 in combination with low dose of $TGF{eta}1$ potentiates the expression of later osteogenic markers osteopontin, osteocalcin and collagen type 1 (COL1a2), while higher concentrations of $TGF{eta}1$ decrease the expression of osteopontin and osteocalcin but not COL1a2. Cell cycle analysis reveals that $TGF{eta}1$ inhibits C3H10T1/2 proliferation in BMP9-induced osteogenesis and restricts the cells in $G_0/G_1$ phase. Our findings strongly suggest that $TGF{eta}1$ may exert a biphasic effect on BMP9-induced osteogenic differentiation of mesenchymal stem cells.