Objective: To investigate the effect of perfluorooctanoic acid (PFOA) on the biological behavior of nasopharyngeal carcinoma cell line HK1 and its potential mechanism of action. Methods: HK1 cells were continuously treated with 50μM concentration PFOA for 10 days. The Cell proliferation ability at 0, 24, 48 and 72 hours was detected by the Cell Counting Kit-8 (CCK-8) method, and the cell migration ability was evaluated by Transwell assay. RNA sequencing (RNA-seq) analysis was performed on HK1 cells treated with PFOA for 10 days to screen for differentially expressed genes (DEGs). The expression levels of key DEGs were verified by quantitative real-time polymerase chain reaction (qPCR) and Western blotting (WB). Functional enrichment analyses of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) were conducted on DEGs. Results: With the extension of culture time, the cell viability of both the control group and the PFOA treatment group gradually increased. At 72 hours, the cell viability of the PFOA treatment group was significantly higher than that of the control group (P<0.05). The Transwell assay showed that the number of migrating cells in the PFOA group was significantly higher than that in the control group (P<0.05). A total of 891 DEGs were screened out by RNA-seq, among which 279 were up-regulated and 612 were down-regulated. The results of qPCR and WB verification showed that the expression levels of key mRNA and protein up-regulating DEGs such as HBEGF, EPHA2, SPRY4, and IL1B were consistent with the sequencing results (P<0.05). Enrichment analysis indicated that DEGs were mainly enriched in Toll-like receptor signaling pathways, cytokine-cytokine receptor interactions, IL-17 signaling pathways, and pathways related to autoimmune diseases, etc. Conclusion: In the HK1 cell model, PFOA can promote the proliferation and migration of nasopharyngeal carcinoma cells. Its effect may be related to the regulation of DEGs such as HBEGF and EPHA2 and inflammation-related signaling pathways, providing preliminary experimental clues for the subsequent exploration of environmental risk factors for nasopharyngeal carcinoma.