Abstract: In order to study the physiological responses of tobacco seedlings to low temperature stress and the change of leaf shape after resuming growth, 7th-leaf stage tobacco seedlings of ‘K326’ were used to measure the physiological indicators of tobacco seedlings at 4℃ and 25℃ for 3, 6, 12, 24 and 48 h. After 4℃ low temperature stress the tobacco seedlings were returned to 25℃ to resume growth. The changes of leaf length and leaf width were measured. The results showed that the relative conductivity, proline content and soluble sugar content increased with the increase of low temperature stress time; the contents of malondialdehyde (MDA) and chlorophyll increased first and then decreased; the activity of catalase (CAT) decreased continuously; the activity of superoxide dismutase (SOD) and peroxidase (POD) had no significant change. After low temperature stress, the leaf length, leaf width and leaf area were significantly inhibited, and the inhibition of leaf width was stronger than that of leaf length, thus increasing the length-width ratio of leaves. After low temperature stress, the length-width ratio of leaves tended to be stable after resuming growth for 16 days. The induction of stress responses of different low temperature stress time was 12 h > 24 h > 6 h > 3 h > 48 h.