Oxygen is toxic effect on Bifidobacteria. The viability of the cells is greatly affected， therefore， hindering its production and application. In order to improve the oxygen tolerance of Bfidobacteria， this experiment used the method of changing the partial pressure of oxygen in liquid medium to carry out the oxygen-tolerant domestication of Bifidobacterium animalis subsp. lactis Probio-M8. The effect of oxygen on Bifidobacterium animalis subsp. lactis Probio-M8 was evaluated by measuring physiological characteristics， oxygen tolerance， and cell membrane fluidity. After oxygen-tolerant if domestication 1 000 generations of growth when the aerobic and anaerobic environment， the pH of culture medium decreased significantly by 0.23 and 0.27（P<0.05）， and the acid production capacity increased significantly. The relative bacterial growth ratio （RBGR） and NADH oxidase activity increased significantly by 0.47 and 31.14 U （P<0.05）， and the oxygen tolerance was significantly enhanced. The unsaturated fatty acid content increased significantly by 17.26% （P<0.05）， and the cell membrane fluidity was enhanced. After oxygen-tolerant domestication， the oxygen tolerance of Bifidobacterium animalis subsp. lactis Probio-M8 was significantly enhanced， which provided ideas for its application and development in food.