Based on published information, the occurrence and development of Alzheimer's disease (AD) are potentially related to gut microbiota changes. Donepezil hydrochloride (DH), which enhances cholinergic activity by blocking acetylcholinesterase (AChE), is one of the first-line drugs for AD treatment approved by the Food and Drug Administration (FDA) of the USA. However, the potential link between the effects of DH on the pathophysiological processes of AD and the gut microbiota remains unclear. In this study, pathological changes in the brain and colon, the activities of superoxide dismutase (SOD) and AChE, and changes in intestinal flora were observed. The results showed that Aβ deposition in the prefrontal cortex and hippocampus of AD mice was significantly decreased, while colonic inflammation was significantly alleviated by DH treatment. Concomitantly, SOD activity was significantly improved, while AChE was significantly reduced after DH administration. In addition, the gut microbiota community composition of AD mice was significantly altered after DH treatment. The relative abundance of Akkermansia in the AD group was 54.8% higher than that in the N group. The relative abundance of Akkermansia was increased by 18.3% and 53.8% in the AD_G group and the N_G group, respectively. Interestingly, Akkermansia showed a potential predictive value and might be a biomarker for AD. Molecular docking revealed the binding mode and major forces between DH and membrane proteins of Akkermansia. The overall results suggest a novel therapeutic mechanism for treating AD and highlight the critical role of gut microbiota in AD pathology.