This study aimed to investigate the expression levels and clinical implications of interferon α inducible protein 27 (IFI27) and the N6-methyladenosine (m6A) regulator Alkylation repair homolog 5 (ALKBH5) in head and neck squamous cell carcinoma (HNSCC). We employed bioinformatics methods to analyze the differential expression of IFI27 in HNSCC and its prognostic implications. Additionally, we explored the pathways and mechanisms associated with its enrichment. Immunohistochemistry (IHC) was used to detect the expression of IFI27 protein in HNSCC and adjacent normal tissues. IFI27 expression was significantly up-regulated in HNSCC (P < 0.001), and was correlated with T stage and tumor differentiation degree. The survival curve indicated that patients with high IFI27 expression had shorter overall survival compared to those with low expression. Furthermore, multivariate analysis confirmed that IFI27 expression is an independent prognostic factor in HNSCC patients. IFI27 is involved in the regulation of type I and type III interferon-mediated responses, Retinoic acid-inducible gene I (RIG-I) -like receptor signaling pathways, and biological processes related to innate immune responses. High IFI27 expression is a potential risk factor for the onset and progression of HNSCC. Additionally, the down-regulation of ALKBH5 may enhance IFI27 expression via the RIG-I/IFN-α axis, further influencing the development of HNSCC.© 2025. The Author(s).