Introduction: Our aim is to reduce the side effects and increase the efficiency of donepezil by formulating donepezil-loaded poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) nanoparticles (NPs) directly targeting amyloid beta (A beta) fibrils in the brain and evaluate behavioral changes in this fibril model of AD.Methods: AD model was developed by intracerebroventricular injection of pre-aggregated beta 25-35 fibrils. Rats were intravenously administered either solvent, donepezil-loaded NPs (15 mu g/kg) or free donepezil (1mg/ kg) 3 times for a week except for naive controls. The effect of treatments on anxiety, motor functions, and cognitive functions was tested by elevated plus maze, locomotor activity, novel object recognition, and Morris's water maze tests, respectively.Results: Accumulation of A beta 25-35 fibrils in brain sections was confirmed. Anxiety-like behavior was observed in the A beta Alzheimer and free donepezil treatment groups while donepezil-loaded NP treatment showed hypo-anxiety-like behavior. Donepezil-loaded NPs were successful in treatment of short-term memory deficit better than free donepezil injection. In Morris's water maze, both donepezil-loaded NPs and free donepezil groups found the platform in shorter time compared to A beta Alzheimer group. In locomotor activity test, both donepezil treated groups moved less than the A beta Alzheimer group and naive controls. After the pharmacological experiments, acetylcholinesterase activity was determined and showed an increase in A beta Alzheimer group compared to controls. Donepezil-loaded NPs inhibited the acetylcholinesterase activity more efficiently than the free donepezil group.Conclusion: Targeting with donepezil-loaded PLGA-b-PEG-NPs increases efficiency, helps to inhibit acetylcholinesterase activity more substantially, improves cognitive decline due to its longer duration of action and destabilizing effect on amyloid fibrils.