Objective To investigate the effect from mechanical stimulation and osteogenic chemical inductor on osteoblastic differentiation markers and formation of calcified nodules in rat bone mesenchymal stem cells (rBMSCs). Method The rBMSC were cultured in medium contained with or without osteogenic chemical inductor. The cyclic biaxial mechanical strain (2%), at a frequency of 1 Hz, was applied to the rBMSCs for periods of 2 hours each time, at intervals of 2 hours, 3 times every day, lasting 3 days and 6 days, respectively. The mRNA expression of alkaline phosphatase (ALP), collagen type I (COL I) and osteocalcin (OCN) were analyzed with real time fluorescent quantitation reverse transcription polymerase chain reaction(qRT-PCR) and formation of calcified nodules were detected with alizarin red staining method. Results The mRNA expression of ALP, COL I and OCN were significantly increased in induced group compared with that in the corresponding uninduced group and calcified nodule was observed in the osteogenic chemical inductor group after 6 days with mechanical stimulation. Conclusions Osteogenic chemical inductor and mechanical stimulation can promote the osteoblastic differentiation of rBMSCs.