Abstract: Melatonin is an effective antioxidant that can promote the growth and development of plants under stress and alleviate stress-induced damage. The growth and development of cotton, an important cash crop in China, is severely impacted by salt stress. As such, here, we explored the regulatory effect of melatonin on the growth and development of cotton under salt stress by soaking ‘Guoxin Cotton No. 9’ seeds in different concentrations of melatonin (0, 0.1, 1, 10, 50, 100, 150 mmol∙L⁻¹) under 150 mmol∙L⁻¹ NaCl. We determined the root morphology (total root length, total surface area, total volume, number of lateral roots, root length, surface area and diameter), seedling height, and dry matter weight; thereafter, the most suitable melatonin concentration, 10 μmol∙L⁻¹, was selected. Then, we measured and analyzed the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), as well as the contents of malondialdehyde (MDA) and soluble sugar in leaves and roots of cotton seedlings and the height, biomass, as well as root morphology indexes under 150 mmol∙L⁻¹ NaCl, 10 μmol∙L⁻¹ melatonin, and 150 mmol∙L⁻¹ NaCl plus 10 μmol∙L⁻¹ melatonin. The results revealed that under salt stress, the height of seedlings decreased, root systems were underdeveloped, dry matter weights decreased, the activities of antioxidant enzymes (SOD, POD, CAT, and APX) decreased, and soluble protein content decreased; however, the MDA content was found to increase when compared to normal, salt stress-free condition. After soaking the seeds in 10 μmol∙L⁻¹ melatonin and 150 mmol∙L⁻¹ NaCl, seedling heights and biomass, total root lengths, number of lateral roots, diameter of taproots, activities of SOD, POD, CAT, APX, and content of soluble sugar all increased, but MDA content decreased in roots and leaves. In the absence of salt stress, soaking the seeds in 10 μmol∙L⁻¹ melatonin did not significantly increase the plant heights, but significantly increased taproot diameters, SOD, POD, CAT, and APX activities, as well as soluble sugar content in the cotton plant roots (P<0.05); however, significantly decreased MDA content (P<0.05). Spearman correlation analysis of 19 indices revealed that the total dry weight of seedlings was significantly and positively correlated with plant height, total root length, main root length, root mean diameter, total lateral root number, SOD, POD, CAT, and APX activities in roots and leaves, and soluble sugar content; however, there was a significant and negative correlation between total dry weight and MDA content in roots and leaves. With these comprehensive analyses, we show that exogenous melatonin could alleviate the damage caused by salt stress in cotton seedlings, promote an increase in plant height and dry matter accumulation, improve the resistance of cotton seedlings to salt stress by promoting lateral root development and thickening of main root, and increase the antioxidant enzyme activity and soluble sugar content. In addition, we reveal that exogenous melatonin can promote the development of cotton seedlings under salt stress-free condition; this provides a theoretical basis for the development and utilization of melatonin as well as the regulation of cotton cultivation.