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Background Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is the third leading cause of cancer-related death worldwide. However, few efficacious treatment options exist, and many patients develop therapy resistance, limiting survival benefit. Chimeric antigen receptor natural killer cell (CAR-NK) therapy shows novel promise in cancer treatment with few toxicities in early development. Therefore, the aim of our project was to screen HCC-specific antigens and cytokines for the design and cytotoxic enhancement of CAR-NK against HCC in vitro and in vivo in our clinically relevant murine hepatocellular carcinoma (HCC) model.Methods Primary NK cells were isolated from wild-type C57BL/6 mice, cultured, and stimulated in vitro with different doses of cytokines, including IL-2, IL-7, IL-15. IL-2 stimulated human NK cells were co-cultured with human HCC cell lines (HepG2 and Huh7 cells) to evaluate NK cell killing ability and response to tumor cell stimulation. Phenotypic changes of co-cultured NK cells were analyzed via flow cytometry and qPCR. Furthermore, cytokine array was applied to test immune regulators in co-cultured media. The efficacy of CAR-NK cells was evaluated in our clinically relevant murine hepatocellular carcinoma (HCC) model.Results IL-2 and IL-15 successfully stimulated primary mouse NK cell activation and maturation in a dose-dependent manner via upregulation of cell surface markers CD11b, CD27, and NK1.1. Phenotypic changes of human NK cells co-cultured with tumor cells showed an upregulation of activation markers CD56, IL-7, and IL-15, in conjunction with the downregulation of KLRg1, an NK cell inhibitor. In addition, cytokine array, qPCR, and flow cytometry revealed that growth differentiation factor 15 (GDF15) functioned as an NK cell inhibitory factor to decrease their cytotoxic activity against tumor cells, and its expression levels in tumor cells was positively associated with inhibitory activity of tumor cells to NK cell activation. Blocking GDF15 improved primary NK cell function ex vivo and in animal models.Conclusions This study confirmed that cytokines IL-2 and IL-15 can successfully stimulate NK activation and maturation, and cancer cell-derived GDF-15 inhibits NK cells function, serving as CAR for improving NK cytotoxic activity to HCC cells.