Abstract： Objective To investigate the influences of hyperbaric oxygen (HBO) on cell cycle and differentiation of rat mesenchymal stem cells(rMSC). Methods One month male Wistar rats were choosed as subjects, rMSCs were separated from femur and cnemis by flushing out with low glucose DMEM. rMSCs of he3rd and 4th generation were purified and exposed to 0.2 Mpa and 0.3 Mpa HBO for one hour. A part of them were digested at once and after 24 hours respectively to examined cell cycle by flow eytometry. And the other part of them were induced by 10 μml 5-azacytidine for two weeks and then the results of differentiation of cardiomyocytes were observed. Results The percentage of cells in the stage of S in the group digested at once after exposed to O. 3 Mpa HBO group decreased obviously compared with the other group and retrieved normal level after 24 h, the cell cycles in the other groups weren't different. 5-azacytidine could induce rMSCs into cardiomyocytes in every group. Tropenin and α-actin which were cadiocyte specificity surface marker were all masculine expresstion in every group, but there was no obviously different among groups. Conclusions Compared with 0.2 Mpa HBO exposed group, there is a more significant effect of 0.3 Mpa HBO on cell cycle, and under this condition, HBO has a quiet effect on differentiation of rMSC.