Objective: To investigate how scopolamine affects border cell coding and spatial memory of mice. Methods: Healthy male 2-month-old C57BL/6 mice were randomly divided into saline control group and scopolamine group. Using morris water maze to evaluate spatial memory ability. Then 32 channel microwire electrodes were implanted into retrosplenial cortex (RSC). After 7 days of recovery, the open field experiment was carried out and the electrical signals were recorded at the same time. Border cell were isolated after analysis. Then 25 μg/kg scopolamine or equivalent volume of normal saline was intraperitoneally injected. After 24 hours, the open field experiment was carried out and the electrical signals were recorded at the same time. The changes of border cells recorded before were analyzed. Results: There were significant differences in the learning curve of water maze experiment between saline control group and scopolamine group (P < 0.05), the time in the target quadrant (P < 0.05) and the number of crossing platform (P < 0.05). After intraperitoneal injection of scopolamine or normal saline for 24 hours, the total distance of mice in open field had no significant difference (P > 0.05); Injection of scopolamine had no significant effect on the firing frequency of border cells in mice (P > 0.05); The earth mover's distance (EMD) was significantly increased after scopolamine injection (P < 0.05), while normal saline injection had no significant effect (P > 0.05). Conclusion: Scopolamine may affect the ability of spatial memory by affecting boundary cell coding in mice.