Hepatocellular carcinoma (HCC) is the world’s leading cause of tumor-related mortalities. Natural killer (NK) cells play a critical role at the first immunological defense line against HCC initiation and progression. NK cell dysfunction is therefore an important mechanism for immune evasion of HCC cells. In the present study using a murine HCC model, we revealed the down-regulation of PR/SET Domain 10 (PRDM10) in hepatic NK cells that were phenotypically and functionally exhausted. PRDM10 silencing diminished the expression of natural killer group 2 member D (NKG2D) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), augmented T cell immunoglobulin and ITIM domain (TIGIT) expression, and decreased the expression of interferon-gamma (IFN-γ), perforin, and granzyme B in normal hepatic NK cells in vitro. Consistently, PRDM10-deficient NK cells exhibited impaired cytotoxicity on target cells. In contrast, PRDM10 overexpression promoted NKG2D and Fas ligand (FasL) expression, reduced CD96 expression, and enhanced transcripts of IFN-γ, perforin, and granzyme B in NK cells in vivo. Moreover, PRDM10 silencing and PRDM10 overexpression down-regulated and up-regulated Eomesodermin (Eomes) expression, respectively. In summary, this study reveals PRDM10 down-regulation as a novel mechanism underlying NK cell dysfunction and identifies PRDM10 as a supporting factor of NK cell function.