Abstract: To explore the inhibitory effect of total glycosides of Cistanche deserticola (TG) on HepG2 cells and its mechanism. In this paper, different concentrations (0, 3.5, 10.5, 21, 31.5, 42 μg/mL) TG were treated 24 h on HepG2 liver cancer cells, and the viability of HepG2 cells was detected using CCK8 assay. Hoechst 33342/PI double staining method and Annexin V-FITC/PI were used to detect HepG2 cells apoptosis. The phenomenon of cell migration was detected by cell migration assay. Meanwhile, cell cycle progression changes were detected by flow cytometry. And the expression of α-fetoprotein (AFP), β-catenin, Dishevelled (Dsh), GSK-3β were detected by Western blot. The results showed that TG could reduce the proliferation of HepG2 cells in a concentration dependent manner, with only 31.04% cell viability when TG was 42 μg/mL. In addition, TG could damage the cell structure and induce cell apoptosis, and the apoptosis rate could be as high as 32.44% by AV/PI detection. Moreover, TG could also promote cell necrosis, and limit cell migration. There were significant differences between the treated group and the control group (P<0.05 or P<0.01). Meanwhile, high concentration of TG could arrest HepG2 cells in S and G₂/M phase. Finally, compared with the control group, the relative expression of β-catenin and Dsh decreased, while GSK-3β increased in the TG treated groups. In conclusion, TG could inhibit HepG2 cell proliferation by affecting cell cycle progression, promoting apoptosis, and limiting cell migration. The mechanism might be through Wnt/β-catenin signaling pathway, which activated GSK-3β to degrade β-catenin to achieve liver cancer inhibition.