Melatonin (MT) regulates several physiological activities in plants. However, information on how MT regulates soybean growth under low-temperature (LT) stress is lacking. To better understand how MT promotes plant growth and development under LT stress, we designed this study to evaluate the role of MT pretreatment on soybean seedlings exposed to LT stress. Our results showed that LT stress increased oxidative damage by increasing reactive oxygen species (ROS) accumulation, which affected the growth and development of soybean seedlings. However, the application of 5 µmol L-1 MT significantly decreased the oxidative damage by increasing plant mineral element concentrations and the transcript abundance of antioxidant related genes, which enhanced the decrease in ROS accumulation. These results collectively suggest the involvement of MT in improving LT stress tolerance of soybean seedlings by mediating plant mineral elements and the expression of genes involved in the antioxidant pathway.