Objective To study morphological and biological senescence changes induced by D-galactose in the cultured rat mesenchymal stem cells. Methods After 3rd generations cultured in the DMEM-F12, MSCs were changed into DMEMF12 medium containing 8 g/L D-galatose and cultured to the 6th generations as the inducement group. The comparison were the 6thgenerations which was cultured in the DMEM-F12 medium all along, and then indentified by surface wave. Using flow cytometer to check the comparisons cell cycle change after swing in with 8 g/L D-galatose within the 4 days. In the first 7daysto draw the growth curve to the two groups. Optical and electronic microscope were used to identify the influences of characteristic morphological of mesenchymal stem cells of the two groups, the influences of biological markers were identified by single cell gel electrophoresis and β-galactose dye. Results After treatment with D-galactose, the mesenchymal stem cells displayed morphological and biological changes in the cell senescence with the senescent characteristic morphological markers; 85% of the cells were X-gal dye masculine, and the singal cell gel electrophoresis showed DNA damnification. The flow cytometry showed that 90% of the cells stayed in G 0/G 1, but the cells in S and G 2/M almost disappeared.However, the cells in the control group had no such DNA damages. Conclusion D-galactose can induce senescence of the mesenchymal stem cells, and 8 g/L is the best concentration to do so. This study has provided a good model forthe research of the mesenchymal stem cells senescence.