〔Abstract〕 To investigate the regulatory role of ABCG5 in head and neck squamous cell carcinoma. Methods Immunohistochemical staining was used to detect the expression of ABCG5 in 105 patients with hypopha- ryngeal carcinoma , and the relationship between the expression and the long-term survival time of the patients was analyzed . Immunowestern blotting and flow cytometry were used to detect the expression of ABCG5 in head and neck squamous cell carcinoma cell lines . Flow sorting was employed to investigate the differences in proliferation and metastasis , spheroidization ability , and expression of stem cell molecules between ABCG5 positive and negative cells . si RNA interference was used to further demonstrate whether ABCG5 affects the malignant phenotype . Re- sults ABCG5 was associated with poor prognosis in patients with hypopharyngeal carcinoma. ABCG5 was enriched in stem cells of head and neck squamous cell carcinoma. ABCG5-positive cells had stronger proliferation ( P < 0. 001), metastasis (P < 0. 01), and spheroid formation abilities (P < 0. 001) than negative cells . The expression levels of some stem cell molecules (SOX2 、NANOG、SOX9 、OCT4 、CD44) in ABCG5-positive cells were higher than those in ABCG5-negative cells . The expression of epithelial cell protein E-cadherin was lower in ABCG5-positive cells than that in ABCG5-negative cells , and the expression of interstitial cell proteins N-cadherin , Slug , Snail 1 , and Vimentin were higher (P < 0. 01) . Moreover , interfering with ABCG5 expression significantly inhibited tumor cell spheroid formation (P < 0. 001), as well as the expression of proteins related to cancer stem cells and epitheli- al-mesenchymal transition . Conclusion ABCG5 has a potential biological role in maintaining the function of head and neck squamous cell carcinoma stem cells .