Hepatocellular carcinoma (HCC) is the most prevalent form of primary liver cancer and ranks as the second leading cause of cancer-related mortality globally. Despite advances in current HCC treatment, it remains a malignancy with poor prognosis. Therefore, developing novel treatment options for patients with HCC is urgently needed. Chimeric antigen receptor (CAR)-modified natural killer (NK) cells have shown potent anti-tumor effects, making them as a promising immunotherapy strategy for cancer treatment. Glypican-3 (GPC3), a cell surface oncofetal glycoprotein, is highly expressed in most HCC tissues, but not in normal tissues, and functions as a key driver of carcinogenesis. Given its high expression level on the cell surface, GPC3 is considered as an attractive immunotherapy target for HCC. In this study, two GPC3-specific CAR-NK cells, NK92MI/NH3 and NK92MI/HS20, were established using NK92MI cells, a modified IL-2-independent NK cell line. These cell lines were engineered with third generation GPC3-specific CAR, and their activities were subsequently evaluated in the treatment of HCC. We found that NK92MI/NH3 cells, rather than NK92MI/HS20 cells, exhibited a significant cytotoxicity effect against GPC3 ⁺ HepG2 cells in vitro and efficiently suppressed tumor growth in a xenograft model using NSG mice. In addition, irradiated NK92MI/NH3 cells displayed similar anti-tumor efficacy to unirradiated NK92MI/NH3 cells. Furthermore, we observed that NK92MI/NH3 cells showed higher killing activity against the GPC3 isoform 2 overexpression cell line (SK-Hep1-v2) than those with GPC3 isoform 1 overexpression cell line (SK-Hep1-v1) both in vitro and in vivo. This suggest that the presence of different GPC3 isoforms in HCC may impact the cytotoxicity activity of NK92MI/NH3 cells and potentially influence therapeutic outcomes. These findings highlight the effective anti-HCC effects of NK92MI/HN3 cells, as well as the underlying therapy resistance, suggesting their potential as a promising therapy for HCC.