Cancer stem cells (CSCs) have been characterized to be responsible for multidrug resistance, metastasis, recurrence, and immunosuppressive in head and neck squamous cell carcinoma (HNSCC). However, the diversity of CSCs remains to be investigated. In this study, we aimed to determine the heterogeneity of CSCs and its effect on the formation of tumor microenvironment (TME).We depicted the landscape of HNSCC transcriptome profile by single-cell RNA-sequencing analysis of 20 HNSCC tissues from public databases, to reveal the Cell components, trajectory changes, signaling network, malignancy status and functional enrichment of CSCs within tumors.Immune checkpoint molecules CD276, LILRB2, CD47 were significantly upregulated in CSCs, enabling host antitumor response to be weakened or damaged. Notably, naive CSCs were divided to 2 different types of cells with different functions, exhibiting functional diversity. In addition, CSCs underwent self-renewal and tumor metastasis activity through WNT and ncWNT signaling. Among them, Regulon regulators (IRF1_394g, IRF7_160g, NFKB1_12g, NFKB2_33g and STAT1_356g) were activated in subgroups 2 and 3, suggesting their pivotal roles in the inflammatory response process in tumors. Among all CSCs, naive CSCs appear to be the most malignant resulting in a worse prognosis.Our study reveals the major signal transduction and biological function of CSCs during HNSCC progression, highlighting the heterogeneity of CSCs and their underlying mechanisms in the formation of an immunosuppressive TME. Therefore, our study about heterogeneity of CSCs in HNSCC can bring new insights for the treatment of HNSCC.Copyright © 2022 Xiao, Zhang, Zhang, Deng, Zheng, Du, Shen, Yue, Yi, Xiao and Zhao.