Our previous study suggested that melatonin‐mediated neuroprotective effects are related with the activation of autophagy. However, the mechanism of melatonin‐mediated autophagic activation in prion‐mediated mitochondrial damage is not reported. Alpha‐7 nicotinic acetylcholine receptors (α7nAchR) is a member of nicotinic acetylcholine receptors, and α7nAchR activation regulates via melatonin. Thus, we hypothesized that melatonin‐mediated neuroprotective effect related with to autophagy pathway as a result of α7nAchR regulation. Inactivation of α7nAchR inhibited melatonin‐mediated autophagic activation and protective effect against prion‐mediated mitochondrial neurotoxicity. Also, knockdown of ATG5 blocked the melatonin‐mediated neuroprotection and did not influence to the activation of α7nAchR caused by melatonin. This report is the first study demonstrating that melatonin‐mediated autophagic activation regulates via modulation of α7nAchR signals, and upregulation of α7nAchR signals induced by melatonin plays a pivotal role in neuroprotection of prion‐mediated mitochondrial neurotoxicity. Our results suggested that regulator of α7 nAChR signals including melatonin may have used for neuroprotective strategies for the neurodegenerative disorders including prion diseases.