In our previous reports, BMP9 has shown potent function to induce osteogenic differentiation of mesenchymal stem cells, however, the underlying molecular mechanism of BMP9-induced osteogenesis is needed to be deep explored. In this study, BMP9 was introduced into mesenchymal stem cells by recombinant adenoviruses protocol, then, in vitro and in vivo assays were conducted to evidence whether BMP9 can regulate osteogenic differentiation of mesenchymal stem cells through ERK1/2 kinase pathway. The results showed that BMP9 can activate ERK1/2 kinase through increase the phosphorylated form of ERK1/2 kinase. ERK1/2 kinase inhibitor PD98059 can increase the ALP activity, OPN expression and calcium deposition of C3H10T1/2 cells induced by BMP9. Furthermore, PD98059 also led to enhancement of Runx2 activity and activation of canonical Smad pathway stimulated by BMP9. When ERK1/2 kinase was silenced by RNA interference, BMP9-activated Smad pathway was further enhanced. Moreover, ALP activity, calcium deposition and in invo ectopic bone formation were accordingly increased along with knockdown of ERK1/2 kinase. Taken together, those results intensively suggested that BMP9 can activate ERK1/2 kinase, and inhibition of ERK1/2 kinase can enhance BMP9-induced osteogenic differentiation of mesenchymal stem cells. ERK1/2 are highly capable of negatively regulate BMP9-induced osteoblastic differentiation of mesenchymal stem cells.