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The late spring cold spell severely affects the growth of litchi flower buds. Melatonin, as a signaling molecule, can enhance the plant’s ability to resist abiotic stress by regulating multiple physiological processes. However, there are few studies on the function of melatonin in litchi under cold stress. In the present study, 100 μM of melatonin was selected based on the ABA content in litchi seedlings. To identify genes potentially involved in melatonin and cold stress conditions in litchi, four RNA-seq libraries of litchi leaves under melatonin and cold conditions were constructed. In total, 6.4–8.5 Gb of trimmed bases were generated in each library. Thirty-five genes were randomly selected for qRT-PCR analysis. The results showed a strong positive correlation between the data from qRT-PCR and RNA-seq. A total of 4590 differentially expressed genes (DEGs) were identified in the treatment of melatonin (1845) and melatonin in cold condition (2745). The expression of several genes belonging to starch and sucrose metabolism, plant hormones (auxin, ABA), MAPK, and alpha-linolenic acid metabolism pathways were differentially expressed. The enhanced carbohydrate metabolism might lead to litchi seedlings treated with melatonin to produce more metabolic energy. Abscisic acid can improve cold tolerance. Collectively, our results reveal that pretreatment with melatonin (100 μM) protects litchi seedlings from cold stress through plant hormones and carbohydrate metabolism and provides potential genes for future research.