Neferine is the major alkaloid compound isolated from the seed embryos of lotus. Neferine has many pharmacological effects, such as anti-inflammatory, antioxidative stress, and antiapoptotic effects, and it maintains autophagic balance. The purpose of this study was to explore the mechanism by which neferine attenuates autophagy after permanent cerebral ischemia in rats. We performed permanent cerebral ischemia in rats by middle cerebral artery occlusion (pMCAO) for 12 h with or without administration of neferine or nimodipine, a calcium (Ca2+) channel blocker. Neuroprotective effects were determined by evaluating the infarct volume and neurological deficits. Autophagy and its signaling pathway were determined by evaluating the expression of phosphorylated AMP-activated protein kinase alpha (AMPK alpha), phosphorylated mammalian target of rapamycin (mTOR), beclin-1, microtubule-associated protein 1A/1B-light chain 3 class II (LC3-II), and p62 by western blotting. Autophagosomes were evaluated by transmission electron microscopy. Neferine treatment significantly reduced infarct volumes and improved neurological deficits. Neferine significantly attenuated the upregulation of autophagy-associated proteins such as LC3-II, beclin-1, and p62 as well as autophagosome formation, all of which were induced by pMCAO. Neferine exerted remarkable protection against cerebral ischemia, possibly via the regulation of autophagy mediated by the Ca2+-dependent AMPK/mTOR pathway.