Water management plays an important role in crop-stress tolerance; however, its effect on the cold tolerance of rice seedlings is unclear. Therefore, we set four different water contents (25%, 45%, 65% and 85%) in the substrate with Liangyou 6326 as the experimental material to study the influence of different water contents on the cold tolerance of rice seedlings. The results showed that in the control temperature treatment, length and number of white roots, dry matter weight, stem base width and strong seedling index first increased and then decreased with decreasing water content, and the seedlings were more robust when the water content was 45%. In the cold treatment, the survival rate and strong seedling index first increased and then decreased with decreasing water content, and the cold tolerance of seedlings was the best when the water content was 45%. The mechanism of improving the cold tolerance is that moderate water deficit improved the antioxidant enzyme activity, reduced the content of malondialdehyde and improved root activity and leaf photosynthetic performance. Correlation analysis showed that the survival rate of seedlings was significantly positively correlated with root activity, antioxidant enzyme activity and seedling quality in the control temperature and cold treatment. The above results showed that soil water played an important role in regulating the cold tolerance of rice. Reasonable water management could not only cultivate high-quality seedlings but also improve the cold tolerance of rice seedlings, helping to guarantee high and stable rice yields.