Background: Head and neck cancer (HNC) is the sixth most common cancer globally, and it is an aggressive malignancy with high morbidity and mortality [1-3]. Every year, 650,000 people are diagnosed with, and about 350,000 people die from, head and neck cancer [3]. Currently, 40,000 new cases and 12,460 deaths are reported in the United States annually [4]. Therefore, it is important to investigate the underlying molecular mechanisms of head and neck carcinogenesis to save the lives of HNC patients.Squalene epoxidase (also called squalene monooxygenase, SQLE, or SM) is an enzyme that uses NADPH and molecular oxygen to oxidize squalene to 2,3-oxidosqualene (squalene epoxide). Squalene epoxidase catalyzes the first oxygenation step in sterol biosynthesis and is thought to be one of the rate-limiting enzymes in this pathway. In cancer biology, SQLE is involved in human esophageal squamous cell carcinoma, lung squamous cell carcinoma, breast cancer, and leukemia [5-8]. SQLE has also recently been linked to nasopharyngeal cancer, but no study to date has described the role of SQLE in HNSCC [9]. Based on our TCGA and GEO database analysis, SQLE was found to be overexpressed in HNSCC tissues when compared to adjacent normal tissues (P < 0.0001). Besides, a significantly worse overall survival rate was observed in the HNSCC patients with high SQLE gene expression than those with low SQLE gene expression (P = 0.001). Thus, we hypothesize that SQLE may play an important role in HNSCC, and it is important to investigate the cellular and molecular mechanisms of SQLE in HNSCC. Objectives: This study aims to identify the role and regulatory mechanisms of SQLE in HNSCC. Considering the oncogenicity of SQLE in other types of cancers, a link between SQLE and HNSCC is worthy of further research. Methods: Phenotypic studies were conducted in two HNSCC cell lines (UMSCC1 and UMSCC23) with knockdown of SQLE. Western blotting was used to quantify protein expression levels. MTT, cell colony formation, EdU, migration, and invasion assays were utilized to assess the phenotype of cancer cells. RNA sequencing was used to analyze the global gene expression in cancer cells. Results: Based on the statistical analysis of existing TCGA and GEO datasets, we found that SQLE may play an important role in HNSCC. Therefore, we aimed to study the function of SQLE in HNSCC cell lines by knocking down the SQLE gene and assessing the altered phenotypes of the cancer cells. Knockdown of SQLE in UMSCC1 and UMSCC23 cells led to significantly decreased proliferation, migration, and invasion potential. Considering that PD-L1, EGF, and FGF are therapeutically important in HNSCC, we also investigated their relationships with SQLE. However, the expression level of PD-L1 did not change after SQLE knockdown. Neither EGF nor FGF altered the expression of SQLE in HNSCC. In order to find potential downstream targets of SQLE, we performed RNA sequencing analysis of UMSCC1 cells with SQLE knockdown. In total, 2,994 genes were differentially expressed following SQLE knockdown, including 1,527 up-regulated and 1,467 down-regulated genes. Signaling pathways were found to be significantly altered by SQLE knockdowns, such as MAPK/ERK signaling, mTOR signaling, and cell cycle pathways. Conclusions: This study has demonstrated that SQLE plays a vital role in HNSCC progression by promoting cancer cell growth, migration, and invasion. RNA sequencing analysis indicates that SQLE may participate in the regulation of HNSCC through interacting with multiple critical regulatory pathways. These findings suggest that SQLE may be an essential target for clinical applications in HNSCC.