Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation of mesenchymal stem cells, the molecular mechanism involved remains to be fully elucidated. In this study, we showed that BMP9 simultaneously promotes the activation of Smad1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. Knockdown of Smad4 with RNA interference reduced nuclear translocation of Smad1/5/8, and disrupted BMP9-induced osteogenic differentiation. BMP9-induced osteogenic differentiation was blocked by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. SB203580 decreased BMP9-activated Smads singling, and yet PD98059 stimulated Smads singling in C3H10T1/2 cells. The effects of inhibitor were reproduced with adenovirus expressing siRNA targeted p38 and ERK1/2, respectively. Taken together, our findings revealed that Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation. Also, it is noteworthy that p38 and ERK1/2 may play opposing regulatory roles in mediating BMP9-induced osteogenic differentiation of C3H10T1/2 cells.