AbstractHuman papillomavirus (HPV) is a major causative factor of head and neck squamous cell carcinoma (HNSCC), and the incidence of HPV‐associated HNSCC is increasing. The role of tumor microenvironment in viral infection and metastasis needs to be explored further. We studied the molecular characteristics of primary tumors (PTs) and lymph node metastatic tumors (LNMTs) by stratifying them based on their HPV status. Eight samples for single‐cell RNA profiling and six samples for spatial transcriptomics (ST), composed of matched primary tumors (PT) and lymph node metastases (LNMT), were collected from both HPV‐negative (HPV−) and HPV‐positive (HPV+) patients. Using the 10x Genomics Visium platform, integrative analyses with single‐cell RNA sequencing were performed. Intracellular and intercellular alterations were analyzed, and the findings were confirmed using experimental validation and publicly available data set. The HPV+ tissues were composed of a substantial amount of lymphoid cells regardless of the presence or absence of metastasis, whereas the HPV− tissue exhibited remarkable changes in the number of macrophages and plasma cells, particularly in the LNMT. From both single‐cell RNA and ST data set, we discovered a central gene, pyruvate kinase muscle isoform 1/2 (PKM2), which is closely associated with the stemness of cancer stem cell‐like populations in LNMT of HPV− tissue. The consistent expression was observed in HPV− HNSCC cell line and the knockdown of PKM2 weakened spheroid formation ability. Furthermore, we found an ectopic lymphoid structure morphology and clinical effects of the structure in ST slide of the HPV+ patients and verified their presence in tumor tissue using immunohistochemistry. Finally, the ephrin‐A (EPHA2) pathway was detected as important signals in angiogenesis for HPV− patients from single‐cell RNA and ST profiles, and knockdown of EPHA2 declined the cell migration. Our study described the distinct cellular composition and molecular alterations in primary and metastatic sites in HNSCC patients based on their HPV status. These results provide insights into HNSCC biology in the context of HPV infection and its potential clinical implications.