Recurrence and metastasis are the major causes of mortality in head and neck squamous cell carcinoma (HNSCC). It is suggested that cancer stem cells (CSCs) play pivotal roles in recurrence and metastasis. Thus, a greater understanding of the mechanisms of CSC regulation may provide opportunities to develop novel therapies for improving survival by controlling recurrence or metastasis. Here, we report that overexpression of the gene encoding the catalytic subunit of PI3K (PIK3CA), the most frequently amplified oncogene in HNSCC, promotes epithelial‐to‐mesenchymal transition and enriches the CSC population. However, PIK3CA is not required to maintain these traits and inhibition of the phosphatidylinositol 3‐kinase (PI3K) signaling pathway paradoxically promotes CSC population. Molecular analysis revealed that overexpression of PIK3CA activates multiple receptor tyrosine kinases (RTKs), in which ephrin receptors (Ephs), tropomyosin receptor kinases (TRK) and mast/stem cell growth factor receptor (c‐Kit) contribute to maintain CSC population. Accordingly, simultaneous inhibition of these RTKs using a multi‐kinase inhibitor ponatinib has a superior effect at eliminating the CSC population and reduces metastasis of PIK3CA‐overexpressing HNSCC cells. Our result suggests that co‐targeting of Ephs, TRKs and the c‐Kit pathway may be effective at eliminating the PI3K‐independent CSC population, thereby providing potential targets for future development of a novel anti‐CSC therapeutic approach for HNSCC patients, particularly for patients with PIK3CA amplification.