Objective To explore the effect of hepatitis C virus (HCV) infection on acetylcholinesterase (AchE) expression level and the effect of AchE activity on the infection of HCV. Methods Human hepatoma cell line Huh7 cells were infected with cell culture-derived HCV (HCVcc). AchE expression level was analyzed using Western blotting and real-time quantitative PCR (qPCR). AchE enzyme activity was assayed using AchE activity detection kit. Huh7 cells were treated with AchE inhibitor, donepezil or itopride, together with HCVcc infection, and then the HCV infection level of Huh7 cells was detected by Western blotting and immunofluorescence. Huh7 cells were transfected with small interfering RNA (siRNA) targeting to AchE gene, followed by infecting with HCVcc, and then the expression levels of AchE and HCV infection were analyzed by Western blotting and immunofluorescence. Results After 60 h of HCVcc infection, the expression level of AchE protein in Huh7 cells and the AchE activity were significantly increased (both P<0.01). AchE inhibitors significantly inhibited HCV infection of Huh7 cells in a concentration-dependent manner (P<0.01). After knocking down AchE with siRNA, HCV infection was significantly inhibited (P<0.01). Conclusion HCV infection of Huh7 cells can up-regulate the expression level of AchE and enhance the activity of AchE. The increase of AchE expression and activity can promote HCV infection, indicating that AchE plays a positive feedback enhancement effect on HCV infection of Huh7 cells.