|Aims: The major malignant phenotypes of cancer, including recurrence, metastasis, and chemoresistance, are related to the presence of cancer stem cells (CSCs). Epithelial Cell Adhesion Molecule (EpCAM) is a type 1 transmembrane protein that is detectable in hepatic stem/progenitor cells and cholangiocytes. Natural killer (NK) cells are the prototype innate lymphoid cells endowed with potent cytolytic function that provide host defense against microbial infection and tumors. However, there are conflicting results: on the cytotoxicity of NK cells against cancer stem cells. In this study, we investigated whether NK cells exhibit enhanced or decreased cytotoxicity against EpCAM+ liver CSCs and the underlying mechanism of the phenomenon. Methods: EpCAMhigh and EpCAMlow Huh-7 cells were sorted by flow cytometry. Human NK cells were isolated by magnetic sorting from peripheral blood mononuclear cells of healthy donors. NK cell and hepatoma cells were co-cultured and cytotoxicity assay was performed. Immunohistochemical staining was performed using human HCC tissues obtained from surgical specimens from the patients who underwent liver resection.Results: Patients with positive EpCAM expression in their tumors showed higher serum α-fetoprotein levels and histological Ki-67 expression. The frequency of early and massive recurrence was higher in patients with positive EpCAM expression in their tumors. Co-culture experiment demonstrated that EpCAMhigh Huh-7 cells resisted NK cell-mediated cytotoxicity. We have screened the potential activating or inhibiting ligands of NK cell receptors, and identified that carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), which is known to inhibit NK-cell-mediated killing, was upregulated on the surface of EpCAMhigh Huh-7 cells. Silencing CEACAM1 restored cytotoxicity of NK cells against EpCAMhigh Huh-7 cells, suggesting that CEACAM1 is responsible for attenuated NK cell-mediated killing of these cells.Conclusions: Overall, our data clearly demonstrate that EpCAMhigh liver CSCs resist NK cell-mediated cytotoxicity by upregulation of cell surface CEACAM1 expression.