Background <p>V-set domain–containing T-cell activation inhibitor 1 (VTCN1) is an immune checkpoint molecule implicated in tumor immune evasion, yet its epigenetic regulation and clinical relevance in head and neck squamous cell carcinoma (HNSCC) remain unclear. Here we systematically investigated the clinical significance and tumor microenvironment (TME) associations of VTCN1 promoter methylation in HNSCC.</p> Methods <p>Three tumor-specific CpG sites within the VTCN1 promoter were identified and integrated into a composite methylation score, which was subsequently used to stratify patients into methylation-high and methylation-low groups. Associations with clinicopathological characteristics and tumor immune microenvironment features were systematically evaluated. In addition, the relationship between VTCN1 promoter methylation status and immunotherapy response was assessed through computational modeling.</p> Results <p>We found that VTCN1 promoter hypomethylation was prevalent in HNSCC tumors and was associated with aggressive clinicopathological features and poorer survival outcomes. Tumors with hypomethylated VTCN1 promoter exhibited an unfavorable immune microenvironment, characterized by reduced immune cell infiltration, cytolytic activity, and stromal components. Moreover, VTCN1 promoter methylation was correlated with IFN-γ–related pathways and immune checkpoint molecules, suggesting an association with T-cell–related immune activity. Notably, patients with a hypermethylated VTCN1 promoter were predicted to exhibit response-related profiles to immunotherapy.</p> Conclusion <p>These findings suggest that VTCN1 promoter methylation may serve as a potential epigenetic biomarker for prognosis, immune infiltration, and response to immunotherapy in HNSCC. Nevertheless, these computational insights remain associative rather than definitive, underscoring the need for further prospective and experimental validation to establish their translational utility.</p>