In addition to Smad pathway, our previous study has shown that BMP9 can induce osteogenic differentiation of mesenchymal stem cells (MSCs) through p38 MAPKs pathway. In this study, we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-indcued osteogenic differentiation of MSCs. BMP9 was introduced into MSCs by recombinant adenoviruses protocol, then, in vitro and in vivo assays were conducted to detect whether BMP9 can induce osteogenic differentiation of MSCs through JNKs kinase pathway. The results showed that BMP9 can activate JNKs kinase through increase the phosphorylated form of JNKs kinase. JNKs kinase inhibitor SP600125 can inhibit ALP activity, OPN and OCN expression, as well as calcium deposition induced by BMP9 in MSCs. Furthermore, SP600125 also led to a decrease in BMP9-induced Runx2 activity and canonical Smad signaling. Moreover, when JNKs kinase was silenced by RNA interference in MSCs, BMP9-induced osteogenic differentiation in vitro and ectopic bone formation in vivo were accordingly inhibited along with knockdown of JNKs. Taken together, those results intensively suggested that BMP9 can induce and regulate osteogenic differentiation of MSCs through activating JNKs kinase pathway.