Although a blockade of acetylcholine esterase has been reported to suppress neuronal cell death induced by exogenous glutamate and beta-amyloid, information is still limited regarding the neuroprotective effects of the acetylcholine esterase inhibitor donepezil. We histologically examined the effects of donepezil on neuronal injury induced by ischemia-reperfusion. Intravenous and intravitreous treatment with donepezil 15 min prior to ischemia dramatically reduced the retinal damage. The protective effect of donepezil in the ganglion cell layer was not affected by mecamylamine, a nicotinic acetylcholine-receptor antagonist, nor scopolamine, a muscarinic acetylcholine-receptor antagonist. The protective effect of donepezil in the inner plexiform layer was reduced not by mecamylamine, but by scopolamine. Neostigmine, a choline-esterase inhibitor, and pilocarpine, a muscarinic acetylcholine-receptor agonist, have protective effects in the inner plexiform layer and the inner nuclear layer. These results suggest that not only the activation of acetylcholine receptors but also a mechanism unrelated to acetylcholine-esterase inhibition contribute to the protective effect of donepezil on the ganglion cells in the ischemic-reperfused rat retina. Donepezil may be useful as a therapeutic drug against retinal diseases that cause neuronal cell death such as glaucoma with high intraocular pressure.