Diabetic patients are more vulnerable to cerebral ischemia-reperfusion (CIR) injury and have a worse prognosis and higher mortality after ischemic stroke than non-diabetic counterparts. Melatonin can exert neuroprotective effects against CIR injury in nondiabetic animal models. However, its effects on diabetic CIR injury and the underlying mechanisms remain unclarified. Herein, we found that melatonin administration improved neurological deficit, cerebral infarct volume, brain edema, and cell viability, reduced mitochondrial swelling, reactive oxygen species generation, and cytoplasmic cytochrome C release, and increased mitochondrial antioxidant enzymes activities, adenosine triphosphate production, and mitochondrial membrane potential in both streptozotocin-induced diabetic mice and high glucose-treated HT22 cells. Importantly, melatonin also activated protein kinase B (Akt) and sirtuin 3 (SIRT3)/superoxide dismutase 2 (SOD2) signaling and upregulated mitochondrial biogenesis-related transcription factors. However, these effects were largely attenuated by LY294002 (a specific Akt signaling blocker) administration. Additionally, 3-TYP (a selective SIRT3 inhibitor) and SIRT3 siRNA inhibited the above protective effects of melatonin as well as the upregulation of SIRT3 and the decrease of SOD2 acetylation but did not affect the p-Akt/Akt ratio. Overall, we demonstrate that melatonin can alleviate CIR injury in diabetic mice by activating Akt-SIRT3-SOD2 signaling and subsequently improving mitochondrial damage.