Objective To investigate the chemosensitization effects of genistein (Gen) combined with 5-fluorouracil (5-FU) on hepatocellular carcinoma in vitro and in vivo.Methods In the in vitro experiments, human hepatocellular carcinoma cell lines (Bel-7404, SMMC-7721 and HepG2) were used in negative control (NC) group, Gen group (10 μmol/L), 5-FU group (2 μmol/L) as well as Gen+5-FU group according to the intervention methods. The effects of Gen combined with 5-FU on the proliferation and apoptosis of hepatocellular carcinoma cells were analyzed by cell counting kit 8 (CCK-8) method and flow cytometry. Bel-7404 cell line-derived xenograft (CDX) and Liver-0117 patient-derived xenograft (PDX) mouse models were constructed in vivo. Then the tumor-bearing mouse models were divided into 4 groups:NC group, Gen group, 5-FU group and Gen+5-FU group. The effects of different treatment methods on tumor growth were observed.Results The proliferation rates of Bel-7404, SMMC-7721 and HepG2 cells in the Gen+5-FU group were significantly lower than those in the Gen group and 5-FU group (all P < 0.01) after 24, 48 and 72 h of treatment; the average apoptosis rates of these cells were significantly higher in the Gen+5-FU group than in the NC group, Gen group and 5-FU group after 72 h of culture (all P < 0.01). The relative tumor volumes (RTVs) of the Bel-7404 CDX tumor-bearing mice in the NC group, Gen (15 mg/kg) group, 5-FU (30 mg/kg) group and Gen (15 mg/kg)+5-FU (20 mg/kg) group 28 d after administration were 15.35±2.92, 17.51±3.11, 12.93±4.88 and 13.67±3.54, respectively, showing no significant difference (F=1.822, P > 0.05); after increasing the dose of Gen to 40 mg/kg for 21 d, the RTV of the Gen (40 mg/kg) group was significantly lower than that of the NC group (9.73±5.85 vs 17.54±5.06, P < 0.05). The RTV of the Liver-0117 PDX tumor-bearing mice in the Gen (15 mg/kg)+5-FU (20 mg/kg) group 18 d after administration was significantly lower than that in the 5-FU (30 mg/kg) group (12.64±0.92 vs 18.76±0.68, P < 0.05), and the ratio of RTV between treatment group and control group was 0.576.Conclusion Gen combined with 5-FU shows good chemosensitization effects in vitro, which are varied on different models of tumor-bearing mice.