U0126 has been reported as a specific inhibitor of the ERK1/2 signaling pathway, which plays a vital role during the osteogenic differentiation of mesenchymal stem cells (MSCs). We report the positive effect of U0126 on the osteogenesis of rat MSCs. We find that U0126 promotes the osteogenic differentiation of rat MSCs as demonstrated by the quantitative real-time polymerase chain reaction for osteogenic markers, alkaline phosphatase activity and calcium nodule formation. Our data indicate that U0126 enhances the BMP/Smad signaling pathway in rat MSCs, while inhibiting the ERK1/2 signaling pathway. Furthermore, Western blot results demonstrate that U0126 increases Smad1/5/8 phosphorylation synergistically with β-glycerophosphate. In addition, U0126 significantly increases the expression of BMP2 during the process of osteogenesis in rat MSCs and the level of phosphorylated Smad1/5/8 is significantly reduced by BMP2 antibody, suggesting that U0126 also promotes the expression of BMP2 to enhance Smad proteins phosphorylation. Thus, we demonstrate a novel function for U0126 in promoting osteogenic differentiation of rat MSCs by the activation of the BMP/Smad signaling pathway.