Acute ischemic stroke is undoubtedly the leading cause of mortality and disability throughout the world, and the efficient treatment is limited so far. Therefore, potentially effective drugs for acute ischemic cerebral damage and subsequent ischemia-reperfusion (I/R) injury are urgently needed. Here, we explored the protective role of melatonin in I/R and investigated the possible molecular mechanism that might affect neuronal autophagy and survival following I/R injury. Our results showed that melatonin can rescue the oxidative stress and cell viability caused by oxygen-glucose deprivation/reoxygenation (OGD/R). Moreover, melatonin treatment significantly increased miR-221 levels in reactive oxygen species (ROS)-dependent manner, thereby mediating the neuroprotective effect of melatonin. Further, we discovered that ATG7 is a target gene of miR-221, indicating that the melatonin-mediated autophagy regulation mechanism is through the miR-221 and ATG7 pathways. Also, we evaluated the more potent combined effect of melatonin and miR-221 on OGD/R SH-SY5Y cells and middle cerebral artery occlusion (MCAO) animals. Together, our study revealed previously unappreciated mechanism of melatonin that mediate neuronal protection through a novel ROS/miR-221/ATG7 axis. The combined application of melatonin and miR-221 is expected to be a potential treatment for inhibiting neuronal damage in stroke patients.