In the previous reports, bone morphogenetic protein 9(BMP9) has shown potent function to induce osteogenic differentiation of mesenchymal stem cells, however, the underlying molecular mechanism of osteogenesis induced by BMP9 is needed to be deep explored. BMP9 was introduced into C3H10T1/2 mesenchymal stem cells by recombinant adenoviruses protocol, then, in vitro and in vivo assays were conducted to evidence whether BMP9 can induce osteogenic differentiation of C3H10T1/2 mesenchymal stem cells through p38 kinase pathway. The results showed that BMP9 can activate p38 kinase through increasing the phosphorylated form of p38 kinase. P38 kinase inhibitor SB203580 can inhibit the ALP activity, OPN expression and calcium deposition of C3H10T1/2 cells induced by BMP9. Furthermore, SB203580 also led to inhibition of canonical Smad pathway activated by BMP9. Moreover, when p38 kinase was silenced by RNA interference in C3H10T1/2 cells, BMP9-induced ALP activity, OPN expression，calcium deposition and in invo ectopic bone formation were accordingly inhibited along with knockdown of p38 kinase. Taken together, those results intensively suggested that BMP9 can induce and regulate osteogenic differentiation of mesenchymal stem cells through activating p38 kinase pathway.