Objective To investigate the expression of FOXO4 in head and neck squamous cell carcinoma (HNSCC) and compare its expression differences among different clinicopathological subtypes, while validating its role in regulating epithelial-mesenchymal transition (EMT). Methods The differential expression of FOXO4 between HNC tissues and normal tissues, as well as its correlation with patient prognosis, was analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) and Ualcan databases. A tissue microarray comprising 117 cases of HNSCC tissues and adjacent normal tissues was utilized to examine the expression of p-FOXO4 based on immunohistochemical results, and its correlation with clinicopathological indicators was compared. Western blot and PCR techniques were performed to detect FOXO4 expression levels in HNC Fadu cells. Stable overexpresssing-FOXO4 cell line-Fadu/FOXO4 and Fadu/Vector (control)—were constructed using DNA transfection technology. Western blot and PCR were used to measure the expression levels of FOXO4, E-Cadherin and Slug in these cell lines. Finally, wound healing assays and Transwell invasion and migration assays were conducted to evaluate the scratch closure distance and the number of invading and migrating cells in Fadu/FOXO4 and Fadu/Vector cells.Results Database analysis revealed that FOXO4 expression was significantly lower in HNC tissues compared to adjacent normal tissues (P<0.01), and its expression was negatively correlated with Slug protein. Immunohistochemical results from the tissue microarray validated this analysis, showing that p-FOXO4 expression was significantly lower in HNC tissues than that in adjacent normal tissues (P<0.01). Additionally, p-FOXO4 expression was lower in HNC patients with tumors larger than 5 cm, lymph node involvement, and distant metastasis (P<0.05).In vitro experiments indicated that FOXO4 overexpression upregulated E-Cadherin expression while downregulated Slug expression in Fadu cells. Furthermore, FOXO4 overexpression inhibited the invasion and migration of HNC cells. Ultimately, combined with database survival analysis, low FOXO4 expression was associated with poor prognosis in HNC patients. Conclusion FOXO4 is lowly expressed in HNC and closely related to various clinicopathological indicators. Overexpression of FOXO4 could inhibit epithelial-mesenchymal transition, invasion, and migration in HNC, and its low expression may indicate poor prognosis in HNC patients