‘Xinluzao 33’ (cold sensitive variety) and ‘Zhongmiansuo 50’ (high cold resistant variety) were selected as test materials in this study.The change of activity of antioxidant enzyme activities and osmotic adjustment substance contents in leaves of seedling were determined under simulating different stress-temperature gradient and time gradient of low temperature stress.The results showed that:(1)The responses to low temperature stress in two cotton cultivars intensified gradually with the decline of temperature and treatment period time,especially 15 ℃ and 10 ℃.(2)With prolonging of stress time,the relative electric conductivity (REC),POD activity,SP and Pro content of cotton leaves generally increased after 15 ℃ and 10 ℃ treatment,the variation trend in activity of POD and APX,content of MDA and SS was increased firstly until 24 h treatment.Under 5 ℃ and 0 ℃ treatment,REC,the activities of SOD and POD,the contents of MDA,SP and SS were increased significantly,the variation trend of APX and Pro increased to a certain degree and then decreased.(3)Compared with ‘Xinluzao 33’,the activities of antioxidant enzymes and the contents of osmotic adjustment substances of ‘Zhongmiansuo 50’ rose more steeply.Those results indicated that,under varying degree of low temperature stress,cotton seedlings could start the appropriate adjustment method to maintain the metabolic balance,and to improve resistance by the regulation and adaption of different indicators of antioxidant enzymes and osmotic adjustment substances.High cold resistant varieties could start the response mechanism of scavenging free radicals and osmotic regulation more rapidly and sensitively,which would enhance chilling adaptability.