Objective · To investigate the effects of neuropeptide substance P（SP） on RUNX2 expression as well as cell proliferation in osteoblasts. Methods · Primary cultured murine skull-derived osteoblasts were isolated from C57BL/6J mice；gene expression of RUNX2 in osteoblast was silenced by transfection with siRNA. Osteoblasts were treated with SP，with or without Spantide（specific blocking agents of SP），L703606（specific blocking agents of NK-1 receptor）and RUNX2 siRNA，then the effects of SP on RUNX2 expression and cell proliferation were investigated. Osteoblast proliferation was determined by cell proliferation-toxicity assay kit（CCK-8） assay. RUNX2 expressions at the mRNA and protein levels were analyzed by real-time PCR and Western blotting analysis，respectively. Results · CCK8 assay showed that SP at the concentration of 10-12～10-6 mol/L promoted osteoblasts proliferation, with 10-8 mol/L being the most effective concentration. The expressions of RUNX2 at the mRNA and protein levels were significantly increased after osteoblasts were treated with 10-8 mol/L SP for 48 h. Moreover，Spantide and L703606 inhibited SP-induced enhancement of RUNX2 expression；RUNX2 siRNA reduced the baseline of osteoblast proliferation and further attenuated the promoting effects of SP on osteoblast proliferation. Conclusion · SP induced RUNX2 expression through binding to NK-1 receptor，thus promoting the proliferation of osteoblasts.