Purpose <p>HOXB13 has been shown to act as a tumor promoter in various malignancies; however, its role in nasopharyngeal carcinoma (NPC) remains unexplored. This study aimed to investigate the function of HOXB13 in NPC and elucidate its underlying mechanism to identify novel targets for NPC diagnosis and therapy.</p> Methods <p>HOXB13 expression in NPC was examined through bioinformatic analyses of the TCGA and GEO databases, and the findings were validated using molecular biology techniques. After the transfection of NPC cell lines with siRNA targeting HOXB13 (si-HOXB13), the effects of HOXB13 knockdown on cell proliferation, migration, invasion, and stemness were evaluated. Expression levels of Wnt/β-catenin/SOX2 pathway-related proteins were assessed. In vivo, NPC cells transfected with sh-HOXB13 were injected into nude mice, after which tumor volume and mass were measured, and lung metastases were analyzed using hematoxylin and eosin (H&amp;E) staining.</p> Results <p>HOXB13 knockdown significantly reduced NPC cell viability, suppressed clonogenicity and invasiveness, increased scratch width in wound healing assays, and decreased sphere formation and the proportion of CD133⁺ cells. Additionally, si-HOXB13 significantly downregulated the protein expression of β-catenin, c-Myc, and SOX2. In vivo, the sh-HOXB13 group exhibited reduced tumor mass, volume and lung metastatic nodules compared to the sh-NC group.</p> Conclusion <p>This study demonstrates that HOXB13 facilitates the malignant progression of NPC by regulating the Wnt/β-catenin/SOX2 signaling pathway, suggesting HOXB13 as a potential therapeutic and diagnostic target for NPC, thereby offering a new strategy to improve patient prognosis.</p>