In vitro expansion of mesenchymal stem cell (MSCs) into large number is necessary for their application in cell-based treatment of articular cartilage defects. On the other hand, some studies have indicated that BIO (6-Bromoindirubin-3-Oxime) possesses mitogenic effects on cell culture. The objective of the present study was to examine the effect of BIO on in vitro expansion and chondrogenic differentiation of mouse marrow-derived MSCs. The culture was established using bone marrow tissue obtained from 10 NMRI mice. MSC nature of the isolated cells was verified according to the minimal criteria proposed for MSC. Passaged-3 cells were seeded in 24-well culture plates and treated by 0.05, 0.01, 0.1, 1.0 and 1.5 µM BIO for seven days. The culture without BIO was taken as the control. At the end of cultivation period, the cultures were examined for viable cell number which was then used to calculate population doubling time (PDT). The BIO with higher proliferation-promoting effect was investigated for its chondrogenic effect on MSC culture. There was significantly more viable cells at the cultures treated by 0.1 µM BIO. At this culture the cells tended to double their population in rapid rate (each 43.07 hr) than the cells treated with the other BIO concentrations (p < 0.05). Interestingly treatment of MSC chondrogenic culture with 0.1 µM BIO led to the up-regulation of cartilage specific genes including aggrecan, collagen II and sox9. In conclusion BIO at 0.1 µM could enhance mouse MSC in vitro proliferation as well as their chondrogenic differentiation. These findings would be of great importance for the field of regenerative medicine.