Purpose: Alzheimer’s disease (AD) is a chronic neurodegenerative disorder, with an increasing prevalence rate, mostly related to cholinergic system. According to the difficulties and complications in management of AD, this study was carried out to evaluate the efficacy of grape seed oil (GSO) on scopolamine (Scop) induced Alzheimer’s in male rats. Methods: 64 healthy male Wistar rats received different treatments such as: normal saline (NS), donepezil (Don), Scop and GSO, according to the previously designed protocol. Morris (MWM) was applied for spatial memory tests. Right after the behavioral tests, the brains were removed and the hippocampus was separated for evaluation of acetylcholine levels as well as cell death and neuro inflammation. Results: The results of the test day indicated that the mean Q2 time was increased in both GSO test groups (P<0.05) and Don treated group (P<0.001).The spectrophotometric findings affirm that both GSO co-treatment and post-treatment were effective in augmenting brain acetylcholine levels (P<0.01 and P<0.05 respectively). The microscopic findings of Hamp;E dyed tissues confirmed the above mentioned results for different treatments except for GSO post treatment, in which the viability of cells were very low. Conclusion: The results implied that supplementation of rats with GSO caused a significant augmentation in spatial memory performance as well as acetylcholine levels and cell viability in the presence of Scop. This effect was comparable to that of Don especially when GSO was used as co-treatment.