BACKGROUND AND STUDY AIMS: As a novel immunotherapy, chimeric antigen receptor T (CAR-T) cell technology is successful in treating hematologic malignancies, and exhibits potential benefits in partial solid tumors. Therapies targeting one antigen have some weaknesses, and dual-targeted CAR-T cells may be a better option. Alpha-fetoprotein (AFP) and glypican-3 (GPC3) are both highly expressed in hepatocellular carcinoma (HCC) and serve as important markers. Our study aimed to compare the cytotoxicity effect of AFP and GPC3 dual-targeted CAR-T cells on HCC cells in vitro and its therapeutic effects on a SCID xenograft model with those of single-targeted CAR-T cells. MATERIALS AND METHODS: pLVX lentivirus vectors loaded with AFP CAR, GPC3 CAR, or AFP-GPC3 CAR constructs were transfected into human T lymphocytes. Control T, AFP CAR-T, GPC3 CAR-T, and AFP-GPC3 CAR-T cells were used as effector cells, and HLE (AFP RESULTS: AFP and GPC3 were highly expressed in Huh-7 cells. AFP-GPC3 CAR-T cells exerted significant cell-killing effects on the HCC cells that expressed specific targeting antigen molecules (AFP and GPC3). Besides, AFP-GPC3 CAR-T cells better promoted Th cytokine secretion by Huh-7 cells than AFP CAR-T and GPC3 CAR-T cells. In vivo results suggested that AFP-GPC3 CAR-T cells better inhibited the growth of Huh-7 cell (AFP CONCLUSION: AFP and GPC3 dual-targeted CAR-T cells showed better anti-tumor effects in HCC than AFP or GPC3 single-targeted CAR-T cells.