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Recently, it has been reported that growth and differentiation factor-5 (GDF-5) regulates and promotes osteoblastic and osteogenic differentiation. We here compared effects of GDF-5 and BMP-2 on the proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs). Cell proliferation was significantly increased in BMP-2 and GDF-5 treated group compared to those in control group. The ALP activities of osteogenic differentiation group, BMP-2 treated group, and GDF-5 treated group was higher than in control group. The ALP activity of GDF-5 group was similar with that of BMP-2 group. When the Ca activity was compared in all groups, the Ca level of GDF-5 group was highest and was similar with that of BMP-2 group. As a result of Von Kossa staining and RT-PCR, the calcium deposition and osteogenic gene expression were increased in the GDF-5 group than in the osteogenic differentiation group. In the present study, GDF-5 appears to be an effective stimulator of proliferation and osteogenic differentiation of rBM-MSCs. To this regard, GDF-5 may be a useful regulator for promoting the bone regeneration and formation by inducing rBM-MSCs proliferation and osteogenic differentiation.