With the pumpkin variety ‘Yong’ an No.2’ seedlings as the tested material, we adopted the indoor pot experiment, sprayed different concentrations (0, 50, 100, 150, 200 and 300 μmol·L^-1) of melatonin (MT), and then carried out low temperature (10 ℃/7 ℃) stress treatment, to investigate the changes of seedling growth index, relative electric conductivity, antioxidant enzyme activity, osmoregulation substance content and other physiological indexes of stress resistance, and preliminarily explore the physiological mechanism of exogenous melatonin alleviating chilling injury of pumpkin seedlings. The results showed that: (1) under cold stress, pumpkin seedlings showed symptoms of chilling injury such as leaf wilting, water loss, edge chlorosis and discoloration, inward curling, etc. The growth indexes such as plant height, stem diameter, fresh weight, dry weight and strong seedling index and the relative chlorophyll content of leaves were significantly decreased. The relative electric conductivity, MDA content and O₂^-· production rate of leaves were significantly increased by more than 50%, and the activities of superoxide dismutase, peroxidase, catalase, ascorbic acid peroxidase, glutathione reductase, dehydroascorbic acid reductase, soluble sugar, soluble protein and proline were also significantly increased. (2) Compared with the cold stress treatment, the chilling injury symptoms of pumpkin seedling leaves were recovered to different degrees after spraying different concentrations of melatonin on the leaves, and the growth state of 100 μmol·L^-1 MT treatment was the best, and there was no significant difference between the plant morphology and phenotype and the control. The seedling growth index, the relative chlorophyll content of leaves, the activities of six antioxidant enzymes, and the contents of three osmoregulatory substances all increased to varying degrees, and all increased first and then decreased with the increase of MT concentration, and all were the highest under 100 μmol·L^-1 MT treatment. The study found that exogenous melatonin could increase the chlorophyll content of leaves, enhance the activities of antioxidant enzymes, increase the accumulation of osmoregulation substances, significantly reduce the degree of membrane peroxidation caused by low temperature chilling injury, effectively reduce the damage of cold stress on pumpkin seedlings, enhance their ability to resist low temperature, and restore the normal growth of seedlings, and the spraying concentration of 100 μmol·L^-1 had the best effect.