This study investigated the effects of melatonin (MT) treatment on the edible qualities and lignification in loquat fruit during cold storage (4 °C) for 25 days. The results exhibited that 50 μM MT treatment effectively maintained the external quality of loquat fruit by reducing weight loss and firmness. MT treatment suppressed the decline of sucrose while slowing the changes in glucose and fructose contents. MT application to loquat fruit also increased the contents of phenolic compounds (chlorogenic acid and neochlorogenic acid) and organic acids (malic acid, oxalic acid, and tartaric acid), thereby maintaining the flavor and nutritional quality of loquat fruit. Compared to untreated fruit, MT treatment reduced the accumulation of malondialdehyde (MDA) and improved antioxidant capacity in loquat fruit, as indicated by higher ABTS, DPPH, and FRAP. Furthermore, MT treatment heavily regulated lignin biosynthesis-related enzyme activities during cold storage, including phenylalanine ammonia-lyase (PAL), 4-coumarate: coenzyme A ligase (4CL), cinnamate dehydrogenase (CAD), and peroxidase (POD). Our results suggest that MT treatment could be an effective approach to alleviate lignification and maintaining the nutrition quality of loquat fruit during cold storage.