Background and Aims Studies have demonstrated that pristimerin (PT) can regulate the proliferation, apoptosis, migration and angiogenesis of cancer cells through multiple mechanisms. However, its role in hepatocellular carcinoma (HCC) has been less studied. Therefore, this study was conducted to investigate the effect of PT on the biological behavior of HCC cultured in vitro and its impact on the sensitivity of HCC cells to gemcitabine (GEM) chemotherapy, as well as the mechanism.Methods The inhibitory effects of PT and GEM on the proliferation of HCC cell lines (Huh7, SMMC-7721 and HepG2) were detected by CCK-8 assay. The suitable HCC cell line and drug concentrations were selected for subsequent experiments according to the results of CCK-8 assay. HCC cells were exposed to GEM (GEM group), PT (PT group), GEM plus PT (GEM+PT group) or Wnt/β-catenin signaling pathway inhibitor FH535 (FH535 group), respectively, using the purely cultured cells as the control group. Then, the apoptosis, colony formation, migration, invasion and the mRNA expressions of c-myc, cyclin D1 and surviving were detected by flow cytometry, plate cloning test, cell scratch assay, Transwell test and qRT-PCR, respectively. At the same time, Western blot technology was used to detect the protein expressions of β-catenin, GSK-3β, p-GSK-3β, vimentin (Vim), E-cadherin (E-cad) and cleaved caspase 3 (C-caspase-3).Results The proliferation rates of Huh7, SMMC-7721 and HepG2 cells were significantly decreased after exposure to both PT and GEM, with a concentration-dependent manner (all P<0.05). The Huh7 cells were chosen as the study object, and the treatment concentrations of PT, GEM and FH535 used were 2.0 μmol/L, 20 μmol/L and 20 μmol/L, respectively. Compared with control group, the apoptosis rate, the relative protein expressions of E-cad and C-caspase-3 were increased, the number of colony formation, scratch healing rate, the number of invasive cells, the relative mRNA expressions of c-myc, cyclin D1, survivin and protein expressions of β-catenin, p-GSK-3β and Vim were decreased in GEM group, PT group, GEM+PT group (all P<0.05). The above changes in Huh7 cells were increased successively in order of GEM group, PT group, GEM+PT group and FH535 groups (all P<0.05).Conclusion PT has significant inhibitory effect on the malignant biological behavior of HCC cells, and can enhance the sensitivity of HCC cells to GEM. The mechanism may be related to its inhibition of the activation of Wnt/β-catenin pathway.