BACKGROUND: HOXB8 is implicated in various cancers. However, the effect pattern of HOXB8 in head and neck squamous cell carcinoma (HNSCC) remains unclear. METHODS: Open-access transcriptional profiles, clinical information, and mutational data were downloaded from the Cancer Genome Atlas database. R software was used for all analysis based on public data through specific R packages. Western blot and real-time quantitative PCR was used to detect the protein and RNA level of HOXB8, respectively. In vivo and in vitro experiments were conducted to explore the effect of HOXB8 on HNSCC cells. RESULTS: Here, we discovered that HOXB8 was upregulated in HNSCC tissue and associated with worse clinical outcomes (clinical stage and prognosis). Results indicated that HOXB8 was primarily distributed in the nucleoplasm. Results of cell lines indicated that HOXB8 is upregulated in HNSCC cells. Further experiments, both in vitro and in vivo, revealed that the suppression of HOXB8 can markedly curb the proliferation, invasion, and migration capabilities of HNSCC cells. Results of biological enrichment and western blot indicated that HOXB8 can regulate the PI3K/AKT/mTOR and EMT pathways. It also came to our attention that HOXB8 could modulate the tumor microenvironment in HNSCC. We observed that patients with high HOXB8 expression had lower infiltration levels of CD8 + T cells but higher infiltration levels of M2 macrophages. Finally, we developed a prognostic model based on molecules derived from HOXB8 (ADD2, SYT1, PXYLP1, MRPL33). CONCLUSIONS: Our study contributes to the existing knowledge on HOXB8 in HNSCC, which may inform future research directions.