Root bark of Turcz. has been widely used as a traditional medicine and is a well-known anti-inflammatory agent. We isolated limonoid triterpene, obacunone (Obac) from the dried root bark of . Obac has been reported to exhibit varieties of biological activities including anti-inflammatory, anti-cancer, and anti-oxidant effects. This study aimed to investigate the beneficial effects and biological mechanisms of Obac in osteoblast differentiation and bone matrix mineralization. In the present study, Obac at concentrations ranging from 1 to 100 μM showed no proliferation effects in MC3T3-E1. The treatment of Obac (1 and 10 μM) increased wound healing and migration rates in a dose-dependent manner. Alkaline phosphatase (ALP) staining and activity showed that Obac (1 and 10 μM) enhanced early osteoblast differentiation in a dose-dependent manner. Obac also increased late osteoblast differentiation in a dose-dependent manner, as indicated by the mineralized nodule formation of ARS staining. The effects of Obac on osteoblast differentiation was validated by the levels of mRNAs encoding the bone differentiation markers, including , bone sialoprotein (), osteopontin (), and osteocalcin (). Obac increased the expression of bone morphogenetic protein (BMP), and the phosphorylation of smad1/5/8, and the expression of runt-related transcription factor 2 (RUNX2); Obac also inhibited GSK3β and upregulated the protein level of β-catenin in a dose-dependent manner during osteoblast differentiation. Obac-mediated osteoblast differentiation was attenuated by a BMP2 inhibitor, Noggin and a Wnt/β-catenin inhibitor, Dickkopf-1 (Dkk1) with the abolishment of RUNX2 expression and nuclear accumulation by Obac. Taken together, the findings of this study demonstrate that Obac has pharmacological and biological activates to promote osteoblast differentiation and bone mineralization through BMP2, β-catenin, and RUNX2 pathways, and suggest that Obac might be a therapeutic effect for the treatment and prevention of bone diseases such as osteoporosis and periodontitis.