Objective Dysfunction of the cholinergic system is an important pathogenetic feature of Alzheimer’s disease (AD). However, the efficacy of cholinomimetic agents in treating AD is low, which may be related to the complexity of the affected pathways. In this study, we established an animal model of oxytocin receptor (OXTR) gene silencing in the nucleus basalis of Meynert (NBM) in the basal forebrain, and we investigated the effects of silencing on learning and memory behaviors and on the cholinergic pathway. Methods Recombinant adeno-associated virus ( rAAV) containing short hairpin RNA (shRNA) to the OXTR gene (rAAV9-EGFP-Oxtr-shRNA (OXTR-shRNA)) was injected at different dosages into the NBM region of the basal forebrain. Then, 2, 3, 4 and 5 weeks later, EGFP expression was measured to assess the rate of infection. An optimal dose of OXTR-shRNA and infection duration were selected for subsequent experiments. Rats were randomly divided into Sham, negative control shRNA (Con-shRNA) and OXTR-shRNA groups. Morris water maze and light-dark box were used to assess spatial learning and fear memory, respectively. To evaluate the effect of silencing on OXTR protein expression, immunofluorescence staining was used to detect the protein in the NBM region. Acetylcholine (Ach) level and the expression of choline acetyltransferase (ChAT) protein in the cortex were assessed by ELISA and immunofluorescence staining, respectively. Results The effects of OXTR-shRNA infection showed dose-dependency and time-dependency. In the water maze test, escape latency was longer in the OXTR-shRNA group than in the Sham and Con-shRNA groups. During the consolidation stage of the light-dark box test, the latency to enter the dark chamber was shorter in the OXTR-shRNA group than in the Sham and Con-shRNA groups. Immunofluorescence staining showed that OXTR expression was decreased in the NBM in the OXTR-shRNA group. Ach levels and ChAT expression in the cortex were reduced in the OXTR-shRNA group compared with the two other groups. Conclusions An animal model of OXTR gene silencing in the basal forebrain was successfully established in this study. This animal model has phenotypes of learning and memory impairment and cholinergic system damage, and therefore can be used to study diseases associated with cognitive dysfunction.