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Background: Reduced cortical acetylcholinesterase activity, as measured by 11C‐donepezil positron emission tomography (PET), has been reported in patients with isolated rapid eye movement (REM) sleep behavior disorder (iRBD). However, its progression and clinical implications have not been fully investigated. Here, we explored the relationship between longitudinal changes in brain acetylcholinesterase activity and cognitive function in iRBD. Methods: Twelve iRBD patients underwent 11C‐donepezil PET at baseline and after 3 years. PET images were interrogated with statistical parametric mapping (SPM) and a regions of interest (ROI) approach. Clinical progression was assessed with the Movement Disorder Society‐Unified Parkinson's Disease Rating Scale‐Part III (MDS‐UPDRS‐III). Cognitive function was rated using the Mini‐Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA). Results: From baseline to follow‐up, the mean 11C‐donepezil distribution volume ratio (DVR) decreased in the cortex (p = 0.006), thalamus (p = 0.013), and caudate (p = 0.013) ROI. Despite no significant changes in the group mean MMSE or MoCA scores being observed, individually, seven patients showed a decline in their scores on these cognitive tests. Subgroup analysis showed that only the subgroup of patients with a decline in cognitive scores had a significant reduction in mean cortical 11C‐donepezil DVR. Conclusions: Our results show that severity of brain cholinergic dysfunction in iRBD patients increases significantly over 3 years, and those changes are more severe in those with a decline in cognitive test scores. [ABSTRACT FROM AUTHOR]