Background & Aim Chimeric antigen receptor T (CAR-T) cells have shown excellent efficacy in treatment of hematological malignancies, but its applications in solid tumors need further exploration. In this study, we investigated the intratumor injection of CAR-T cells therapy targeting glypican-3 (GPC3)-positive advanced hepatocellular carcinoma (HCC) patients to evaluate its safety and efficacy (ClinicalTrials.gov Identifier: NCT03130712). Methods, Results & Conclusion Two escalating dose levels (DLs) (1 × 105 to 5 × 105 CAR+ T cells/cm3) of CAR-T were applied in 4 advanced HCC patients. Under ultrasound guidance, injections were made from three angles at two sections of the tumor. GPC3-CAR-T cells proliferated in vivo and were detectable in the peripheral blood of patients who had a response. Three of four patients had the grade 2 cytokine-release syndrome at day 12-15 after intratumor injection of CAR-T cells. Other severe adverse events related to CAR-T therapy were not observed. Serum tumor marker alpha fetoprotein (AFP) was not significantly changed. We also observed that the quality of life and self perception of all patients improved significantly. As of right now, 2 of the 4 patients who experienced progressive disease (PD) in previous treatments had stable disease (SD) for more than 12 weeks. One patient had partial response (PR) that showed portal vein tumor thrombus vanished by MRI analysis after 4 weeks and unfortunately his body began to produced anti-GPC3CAR antibody after second and third injected at 6 and 14 weeks, and he had PD at 18 weeks. This clinical trial will continue as planned. This work was supported by the National Nature Science Foundation of China (81672467, 81702773, 81702389).