Background Immune checkpoint inhibitors (ICIs) are therapeutically effective for hepatocellular carcinoma (HCC) but are individually selective. This study examined the role of specific common fragile sites (CFSs) related gene in HCC immunotherapy. Methods We analyzed HCC tissues using next-generation sequencing and flow cytometry via time-of-flight technology. A humanized orthotopic HCC mouse model, an in vitro co-culture system, untargeted metabolomics and a DNA pulldown assay were used to examine the function and mechanism of WWOX in the tumor immune response. Results WWOX was the most upregulated CFS-related gene in HCC patients responsive to ICIs. WWOX deficiency renders HCC resistant to PD-1 treatment in humanized orthotopic HCC mouse model. Macrophage infiltration is increased and CD8 T-cell subset infiltration is decreased in WWOX-deficient HCC patients. HCC-derived oleic acid (OA) promotes macrophage conversion to an immunosuppressive phenotype. Mechanistically, WWOX deficiency promoted OA synthesis primarily via competitive binding of NME2 with KAT1, which promoted acetylation of NME2 at site 31 and inhibited NME2 binding to the SCD5 promoter region. Pharmacological blockade of SCD5 enhanced the antitumor effects of anti-PD-1 therapy. Conclusions WWOX is a key factor for immune escape in HCC patients, which suggests its use as a biomarker for stratified treatment with ICIs in clinical HCC patients.