Collagen loss is one of the major contributor to signs of skin aging such as dryness, roughness, and wrinkle formation, which is closely linked to a decline in the amount of proline produced in mitochondria. Melatonin has been shown to improve several clinical signs of skin aging, while the mechanism is unclear. In our study, we found that mitophagy, proline synthesis key enzyme NADK2 and proline and collagen levels were significantly reduced, while oxidative stress levels increased in aging skin, and melatonin supplementation could effectively up-regulate mitophagy level and restore proline synthesis and further improved skin aging. However, proline supplementation could also exert an anti-aging effect, while it had no effect on the mitochondrial dysfunction. Moreover, our study indicated that melatonin enters the cell by binding to the MT1 receptor and then enters the mitochondria via the PEPT1 transporter to exert its mitochondrial protective effects. This study helps to elucidate the mechanism of mitochondrial dysfunction-induced skin aging, and provides new theoretical guidance for revealing the mechanism of skin aging and rationally utilizing endocrine hormones to improve skin aging, which has a broad application prospect.