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Objective:To investigate the molecular pathway of substance P(SP)to induce osteoblastic differentiation.Methods:Mesenchymal stem cells were isolated and cultured.The cultures were divided into four groups with Group A(control group)cultured without any factors,Group B cultured with SP,Group C cultured with SP and SP receptor neurokinin-1(NK_1)antagonist,and Group D cultured with SP NK_1 antagonist respectively to induce osteoblastic cells differentiation.Osterix gene expression was detected by reverse transcription-polymerase chain reaction(RT-PCR)for three times after 1-2 weeks of cultivation and the results were analyzed by one-way analysis of variance(ANOVA).Results:The log phase of bone marrow stromal cells appeared at 4-6 days.ALP staining revealed that the majority of cells,more than 95%,were positive and small bluepurple granules were found in the cytoplasm.And Group B,treated with SP,showed a higher level of ALP activity than the other three groups.Meanwhile,RT-PCR found that Osterix expression in Group B was obviously up-regulated,compared with other groups.But Osterix expression in Group D had no remarkable differences,compared with the controls.Conclusions:SP can up-regulate Osterix gene expression to stimulate differentiation of mesenchymal stem cells into osteoblastic cells at the final stage.The regulatory effect of SP on Osterix expression was dependant on SP NK_1 receptors.