In the previous reports, BMP9 has shown potent function to induce osteogenesis, but the underlying molecular mechanism of osteogenesis induced by BMP9 is needed to be deeply explored. Dominant negative typeⅡ TGFβ receptors and BMP9 were constructed by following recombinant adenoviruses protocol and co-introduced into target cells. Then the typeⅡ TGFβ receptors required for BMP9-induced osteogenesis was identified and analyzed through in vitro and in vivo assays. It was found that three dominant negative typeⅡ TGFβ receptors, which are dnBMPRⅡ, dnActRⅡ and dnActRⅡB, can not only reduce alkaline phosphatase (ALP) activity induced by BMP9 and calcium deposition, but also repress the activation of Smad signal pathway. Moreover, dnBMPRⅡ, dnActRⅡ and dnActRⅡB also showed to inhibit ectopic bone formation induced by BMP9 in vivo. However, target cells expressed BMPRⅡ and ActRⅡ, but not ActRⅡB. Then, when BMPRⅡ and ActRⅡ were silenced by RNA interference in target cells, luciferase reporter activity and ALP activity induced by BMP9 was accordingly inhibited along with knockdown of BMPRⅡ and ActRⅡ. Taken together, those results intensively suggest that BMPRⅡ and ActRⅡ are the functional typeⅡ TGFβ receptors required for BMP9 induced osteogenesis.