PURPOSE: As a consequence of the natural aging process, the lacrimal glands may become dysfunctional. The present study aimed to investigate the potential role of melatonin (MLT) in the alleviation of age-related lacrimal gland dysfunction and to elucidate the underlying mechanism. METHODS: In this study, lacrimal glands of 2-month-old, 18-month-old, and MLT intraperitoneally injected 18-month-old mice were obtained for immunofluorescence, immunohistochemistry experiments, and Western blotting to detect inflammatory factors and AQP5 expression, and for electron microscopy to detect mitochondrial structure and dense granules. Lacrimal glands from 18-month-old mice were taken for cell culture, and PCR and Western blotting were performed to detect the signaling pathways in which MLT acts. In addition, the human lacrimal gland explant cultures were performed to validate the role of MLT and the SIRT-1/NLRP3 signaling pathways. RESULTS: In this study, we discovered that aging increased the inflammatory response, decreased secretory function, and led to mitochondrial dysregulation in lacrimal gland. Compared with 2-month-old mice, SIRT-1/3/6 gene transcript levels were significantly decreased in 18-month-old mice. MLT reduced inflammatory factors (IL-1β, IL-6, and TNF-α) and increased AQP5 expression via the SIRT-1/NLRP3 signaling pathway in aged lacrimal gland of human and mouse. Furthermore, MLT restored mitochondrial structure and increased dense granules in aged mouse lacrimal gland. In explants of human lacrimal gland, MLT relieved fibrosis. CONCLUSIONS: The present study demonstrated that MLT alleviates age-related lacrimal dysfunction in mice and humans via the SIRT-1/NLRP3 pathway. MLT alleviated the inflammatory response and the decline in the secretory function of the aged lacrimal gland.