Background <p>Head and Neck Squamous Cell Carcinoma (HNSCC) presents significant challenges in terms of treatment and prognosis, highlighting the urgent need for new therapeutic targets and the development of effective targeted therapies to enhance patient outcomes and survival.</p> Methods <p>The expression level of RBMS1 in HNSCC was identified by GEO and TCGA databases through systematic bioinformatics analysis, and further verified in human specimens by quantitative Real-time PCR, Western blot, and immunohistochemistry. The results of CCK-8, colony formation assay, wound healing, Transwell, and tumor formation assays in nude mice showed that RBMS1 promoted the proliferation, migration, and invasion of HNSCC cells. The downstream target genes of RBMS1 were identified in the RBMS1 knockdown and the control groups of TU177 cells using RNA sequencing. HSPA8 was identified as a downstream target gene of RBMS1 in functional in vitro and tumor formation experiments in nude mice.</p> Results <p>Elevated expression levels of RBMS1 in HNSCC were identified using relevant databases and validated in human specimens. In both in vitro and in vivo studies, overexpression of RBMS1 promoted the proliferation, migration, and invasion of HNSCC cells, whereas knockdown of RBMS1 significantly inhibited these processes. RNA sequencing analysis revealed HSPA8 as a downstream target of RBMS1, and rescue experiments confirmed that HSPA8 serves as a crucial intermediary in the regulatory pathway of tumor progression influenced by RBMS1.</p> Conclusions <p>This study suggests that RBMS1 regulates HSPA8 to promote the proliferation, migration, and invasion of HNSCC cells, making it a potential therapeutic target for HNSCC.</p>