--- - |2- Abstract Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer deaths worldwide, especially in male. With poor prognosis, significant portions of patients with HNSCC die due to cancer recurrence and tumor metastasis after chemotherapy and targeted therapies. The HNSCC FaDu cell ectopic expression of Twist, a key transcriptional factor of epithelial–mesenchymal transition (EMT), which triggers EMT and results in the acquisition of a mesenchymal phenotype, was used as the cell model. Our results demonstrated that treatment with newly synthesized 2‐(3‐hydroxyphenyl)‐5‐methylnaphthyridin‐4‐one (CSC‐3436), a flavonoid derivative, elicited changes in its cell morphology, upregulated E‐cadherin messenger RNA and protein expression, downregulated N‐cadherin, vimentin, and CD133 (a marker associated with tumor‐initiating cells) in FaDu‐pCDH‐Twist cells. Moreover, CSC‐3436 exposure reduced B cell‐specific Moloney murine leukemia virus integration site 1 (Bmi1) expression regulated by Twist and further suppressed the direct co‐regulation of E‐cadherin by Twist and Bmi1. Interestingly, CSC‐3436 reduced EMT, cancer stemness, and migration/invasion abilities through the inhibition of the Twist/Bmi1‐Akt/β‐catenin pathway. Most importantly, our findings provided new evidence that CSC‐3436 played a crucial role in therapeutic targeting to Bmi1 and its molecular pathway in HNSCC, and it will be valuable in prognostic prediction and treatment. - Journal of Cellular Physiology, EarlyView.