Objective To study the response of rat bone marrow mesenchymal stem cells(MSCs) to a single period of mechanical strain and expression patterns of transforming growth factor-β(TGF-β) and insulin-like growth factor- Ⅱ(IGF-Ⅱ) after mechanical stretch. Methods Bone marrow MSCs were isolated from SD rats and cultured in vitro. A four-point bending apparatus were used to perform a single period of mechanical strain(2 000 με, 40 min) on MSCs. Cellular proliferation and alkaline phosphatase(ALP) activity of MSCs were examined and gene expression patterns of TGF-β and IGF-Ⅱ were detected by SYBR green quantitative real-time RT-PCR. Results Cell proliferation, ALP activity and expression of TGF-β and IGF-Ⅱ were all significantly up-regulated in stretched MSCs when compared with their controls. The mRNA levels of TGF-β and IGF- Ⅱ got top increase immediately after mechanical loading and increased about 51.44 and 8.92 folds, respectively, when compared with control cells. Expression of TGF-β and IGF-Ⅱ decreased with time and returned to control level at 12 h after mechanical stimulus, despite of a small increase at 6 h. Conclusion The mechanical stretch can promote MSCs proliferation, up-regulate its ALP activity and induce a time-dependent expression increase of TGF-β and IGF-Ⅱ which in turn result in osteogenic differentiation of MSCs. Mechanical stimulus is a key stimulator for osteogenic differentiation of MSCs and vital for bone formation in distraction osteogenesis.