Abstract: We explored the physiological mechanism of Oryza sativa L. seedlings to resist low temperature stress and different physiological mechanisms of cold tolerance between indica O. sativa and japonica O. sativa. Two indica varieties (O. sativa L. subsp. indica Kato) and two japonica varieties (O. sativa L. subsp. japonica Kato) were used as materials. We compared the changes in reactive oxygen species (ROS) metabolism, antioxidant enzyme activities, antioxidant contents, and osmotic adjustment substances in O. sativa seedlings after low temperature stress and normal temperature recovery. Results showed that the content of H₂O₂ in the four varieties increased significantly after low temperature stress. The activities of the five enzymes in the antioxidant enzyme system increased to varying degrees; the activity of superoxide dismutase (SOD) increased significantly, whereas that of glutathione reductase (GR) did not. Glutathione (GSH) and ascorbic acid (AsA) were significantly increased in ‘Minghui 86’. The content of the osmotic adjustment substance proline was not significantly changed in ‘Qianhui 1385’ or ‘Nipponbare’, and the soluble sugar content was not significantly changed in ‘Minghui 86’. After 7 d of recovery, the above physiological indices of the four varieties were restored to pre-treatment levels to varying degrees. Results showed that antioxidant enzyme activity effectively reduced the level of ROS and alleviated the damage caused by cold stress to the cell membrane. During low temperature stress, the cells maintained high osmotic adjustment substances, increased intracellular water potential, and reduced damage caused by plant water loss. Therefore, the timely response of various cold-tolerant physiological substances in the cells and the synergistic and efficient operation of the antioxidant system are important physiological indicators for measuring the cold tolerance of O. sativa.