RPS6KA1 express disorder is associated with many cancers, but the role in head and neck squamous cell carcinoma (HNSCC) and the specific mechanism is still unclear. We used bioinformatics analysis to explore the role of Ribosomal Protein S6 Kinase A1(RPS6KA1) in HNSCC which were predicted to regulate certain pathway and immune microenvironment to increase the risk in HNSCC. Multiple bioinformatics tools based on the EBI, GEO, TCGA databases and clinical samples were used to analyze the expression of RPS6K1 in HNSCC. Western blot (WB) and PCR results confirmed the upregulation of RPS6KA1 in HNSCC tissues. Flow cytometry was used to validate the relationship between RPS6KA1 and immune cell infiltration in the tumor microenvironment. The correlation of RPS6KA1 with the immune environment was further analyzed, and flow cytometry validation was performed in HNSCC samples. Then, Gene Ontology(GO) and Gene Set Enrichment Analysis(GSEA) analysis were used to explore the pathway which could be regulated by RPS6KA1 in HNSCC. And drug sensitivity analysis was used to the access the relationships between RPS6KA1 and drugs therapeutic effects. EBI, TCGA and GEO databases were used to reveal that RPS6KA1 expression was significantly increased in HNSCC, especially in III + IV HNSCC. And it was related to many types of immune cells and immune adjustment factors and positively correlated with tumor immune score and B cells, but had no significant correlation with CD4⁺ and CD8⁺ T cells. Drug sensitivity analysis revealed that RPS6KA1 has certain predictive value. In this research, we indicated that RPS6KA1 is overexpressed and may serve as a potential diagnostic and therapeutic biomarker in HNSCC.