Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury. Although melatonin is known as an effective antioxidant, the mechanism of the protection cannot be explained merely by antioxidation. This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy, since autophagy has been frequently suggested to play a crucial role in neuron survival. To find it out, an ischemia/reperfusion model in N2a cells was established for examinations. The results showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reperfusion onset following ischemia and greatly promoted cell survival, while autophagy blockage by 3-MA led to the shortened N2a cell survival as assessed by MTT, transmission electron microscopy, and laser confocal scanning microscopy. Besides, the protein levels of LC3II and Beclin1 were remarkably increased in ischemia/reperfusion-injured N2a in the presence of melatonin, whereas the expression of p-PKB, key kinase in PI3K/PKB signaling pathway, showed a decrease when compared with untreated subjects as accessed by immunoblotting. Taken together these data suggest that autophagy is possibly one of the mechanisms underlying neuroprotection of melatonin.