BACKGROUND: Most cancers, including head and neck squamous cell carcinoma (HNSCC), frequently exhibit an approximately 80% lack of response to immune checkpoint blockade (ICB) therapy, largely attributed to hypoxia-induced tumor immune suppression. Although hypoxia is known to upregulate PD-L1 expression, the key mechanisms by which it enhances PD-L1 membrane localization and high expression remain elusive. METHODS: We investigated the molecular mechanisms by which hypoxia enhances PD-L1 membrane localization in HNSCC cells. Additionally, we tested the efficacy of combining an anti-PD-1 antibody with the NMT1 inhibitor PCLX-001 in HNSCC xenograft mice and conducted a retrospective clinical study to assess NMT1 as a prognostic biomarker. RESULTS: Our study revealed that hypoxia-inducible factor-1α (HIF1α) upregulates N-myristoyltransferase 1 (NMT1), which mediates the myristoylation of calcineurin B homologous protein 1 (CHP1). Myristoylated CHP1 binds to PD-L1, facilitating its rapid translocation to the cell membrane and increasing PD-L1-mediated immune evasion. The NMT1 inhibitor low-dose PCLX-001 blocks CHP1 myristoylation, disrupting excessive PD-L1 membrane localization and attenuating cancer immune suppression. In HNSCC xenograft mice, administering an anti-PD-1 antibody combined with low-dose PCLX-001 via intratumoral injection significantly improved the treatment response rate and produced synergistic anticancer effects with no significant weight loss. Furthermore, our retrospective clinical study demonstrated that NMT1 protein levels can serve as an independent prognostic biomarker for HNSCC. CONCLUSION: These findings provide robust theoretical support for the translational application of combining NMT1 inhibitors and ICB therapy in cancers under hypoxic conditions. This study introduces a combined cancer therapy strategy named "spatial blockade plus signaling inhibition of PD-L1."