Objective To examine the feasibility of expanding MSCs from the isolated bone marrow mononuclear cells using a rotary bioreactor system. Methods The cells were cultured in a rotary bioreactor with Myelocult? medium with addtion of supplementary factors such as stem cell factor (SCF), interleukin 3 and 6 (IL-3, IL-6). The MSCs were measured and compared the changed of its apparent, hyperplasia and differentiation before and after treated by the bioreactor. Resules After 8 days of culture, total cell numbers, Stro-1(superscript +) CD44(superscript +) CD34(superscript +)MSCs and CD34(superscript +) CD44(superscript +)Stro-1(superscript -) HSCs were increased 9, 29, and 8 folds respectively. The bioreactor-expanded MSCs showed expression of primitive MSCs markers endoglin (SH2) and vimentin, whereas markers associated with lineage differentiation including osteocalcin (osteogenesis), Type Ⅱ collagen (chondrogenesis) and C/EBPa (adipogenesis) were not detected. Colony forming efficiency-fibroblast per day (CFE-F/day) of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocyles and adipocyles, which showed no difference with that of MSCs without treating by Bioreactor. Conclusion It could conclude that the rotary bioreactor with the modified Myelocult? medium reported in this study could be used to rapidly expand MSCs.