Abstract: Objective:To explore the molecular mechanism of abnormal expression of forkhead box O1 (FOXO1) in nasopharyngeal carcinoma (NPC) and its impact on the malignant biological behavior of NPC cells. Methods:GEO dataset was used to analyze FOXO1 expression levels in NPC, and R4.2.1 software was used for gene set enrichment analysis. NPC cell lines 5-8F and HONE1 with overexpressing FOXO1 (FOXO1-5-8F, Ctrl- 5-8F, FOXO1 HONE1, Ctrl-HONE1) were constructed. Cell proliferation, migration, and invasion abilities were assessed through the cell counting kit-8 (CCK-8) assay, scratch assay, and Transwell assay, respectively. The cell cycle was analyzed by using flow cytometry. FOXO1 DNA methylation sequencing by bisulfite gene sequencing technology was performed. Results:The expression of FOXO1 mRNA was down-regulated in NPC cells and tissues. Cell proliferation and migration abilities in the FOXO1 overexpression group were significantly lower than those in the control group (P＜0.05), the number of cell invasion in the blank control group was higher than that in the FOXO1 overexpression group at the same time (P＜0.05), and FOXO1 overexpression inhibited the cell cy- cle (P＜0.05). The promoter region of FOXO1 DNA in NPC tissues was observed to contain a methylation site, with a significantly higher level of methylation compared to non- cancerous tissues (P＜0.05). Conclusion:Down-regulation of FOXO1 transcription via DNA hypermethylation in NPC promotes the proliferation and invasion of NPC cells. FOXO1 may act as a tumor suppressor gene for NPC.