<p>Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous malignancy associated with poor prognostic outcomes. Despite ongoing efforts to identify reliable biomarkers for prognosis, the clinical utility of these markers remains limited owing to the need for further validation and deeper mechanistic insights. In this study, we developed and validated a novel keratinization-related gene signature (KRGS) to predict prognosis and radiation therapy (RT) response in human papillomavirus (HPV)-negative HNSCC using data from The Cancer Genome Atlas (TCGA). The 16-gene KRGS effectively stratified patients with HNSCC into two subgroups with significantly differing survival outcomes. KRGS^low, characterized by low KRGS expression, exhibited poorer survival and reduced sensitivity to RT, while KRGS^high, with high KRGS expression, was associated with more favorable survival outcomes and enhanced RT responsiveness. Functional validation, both in vitro and in vivo, demonstrated that keratinization activation through all-<i>trans</i> retinoic acid (ATRA) treatment upregulated the cornified envelope and sensitized HNSCC cells to RT. The enhanced response to RT was further associated with the upregulation of eight KRGS-related genes and increased expression of involucrin (IVL), a key regulator of terminal differentiation during cornification. Notably, the combination of ATRA and IR reduced radioresistance in HNSCC cells, which was linked to the downregulation of integrin alpha-1 (ITGA1) expression. These findings provide new insights into the role of keratinization in modulating radioresistance and suggest that KRGS-driven activation of keratinization, in combination with RT, may represent a promising therapeutic strategy to overcome resistance in HNSCC.</p>