Background: The proportion of non-alcoholic fatty liver disease (NAFLD) has been increasing as a cause of hepatocellular carcinoma (HCC) worldwide. Natural killer (NK) cells are involved in the first line of immune defense against cancer. NK cell function is regulated by activating and inhibitory NK cell receptors. However, the role of NK cells in the pathogenesis of NAFLD and NAFLD-HCC is still largely unknown. In this study, we aimed to clarify the phenotypic and functional features of NK cells in NAFLD and NAFLD-HCC patients. Methods: We performed mass cytometry (CyTOF) and flow cytometry analysis of NK cells in 33 NAFLD patients (22 chronic hepatitis (CH), 8 liver cirrhosis (LC), 3 with HCC (HCC)) and 9 healthy donors (HDs). We compared surface markers on NK cells in cancerous and non-cancerous intrahepatic lymphocytes (IHLs). We also measured NK cell function in the presence of IL-12 and IL-18. Results: The frequency of NK cells was lower in NAFLD patients compared to HDs. PD-1, CD57, ILT2 were highly expressed, and Siglec-7, NKp30, NKp46 were downregulated on NK cells from NAFLD patients, compared with those of HDs. In NAFLD patients, Siglec-7 levels on NK cells were negatively correlated with PD-1 and CD57, and positively correlated with NKp30 and NKp46. The other inhibitory markers (NKG2A, KIR3DL1 and KIRDL2/L3), activating markers (CD69 and NKG2D) and checkpoint markers (Tim-3 and TIGIT) were comparable between NAFLD patients and HDs. PD-1 and CD57 expression levels on NK cells were also significantly upregulated in NAFLD-HCC patients than those in HDs. CD57 was rarely expressed on NK cells in non-cancerous IHLs, on the other hand, highly expressed in cancerous IHLs. The IFNγ production and CD107a expression on NK cells were also decreased in NAFLD patients. PD-1⁺CD57⁺Siglec-7⁻ NK cells were observed in NAFLD patients, rarely in HDs. PD-1⁺CD57⁺Siglec-7⁻ NK cells were functionally impaired compared to other NK subsets. Conclusions: In patients with NAFLD, NK cells are reduced and functionally impaired, the reason of which may be the increased proportion of dysfunctional PD-1⁺CD57⁺Siglec-7⁻ NK subset, and dysfunctional NK cells might be related to impairment of surveillance for HCC.