Mangoes often suffer from low temperature-induced chilling injury (CI) during postharvest cold storage.  Therefore, advanced techniques are crucial and in high demand to solve the chilling stress of mango fruit for a higher value.  This study addresses chilling stress modulation by investigating the effects of melatonin treatment on CI, proline metabolism, and related gene expressions of ‘Keitt’ mango during cold storage after dipped in 0 (control), 0.1 (MT1), and 0.2 mmol L^–1 (MT2) melatonin solution for 30 min.  The results revealed that melatonin treatment in MT1 significantly reduced CI development and increased proline content in mango fruit during cold storage compared to the control.  These changes were along with increases in the activity of critical enzymes as well as the expression of encoding genes involved in proline biosynthesis, such as pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), ornithine D-aminotransferase (OAT), P5CS2, P5CR2, and OAT3.  Additionally, proline dehydrogenase (PDH) activity and the expression of the PDH3 gene associated with proline dehydrogenation were lower in MT1-treated mangoes than the controlled group.  Thus, melatonin treatment has regulated proline metabolism resulting in the accumulation of proline, subsequently contributing to enhancing the chilling tolerance of ‘Keitt’ mango fruit.