Head and neck squamous cell carcinoma (HNSCC) remains a prevalent and lethal malignancy, with a five-year survival rate of just 50% for cases of locally advanced disease. Chromosomal aberrations, particularly the deletion of the short arm of chromosome 3 (3p), have been strongly associated with poor prognosis and more aggressive tumor phenotypes. The tumor microenvironment (TME) plays a pivotal role in tumor progression and resistance to therapy. This study aims to elucidate the impact of 3p deletion on the TME, immune cell infiltration, and treatment response in HNSCC, to identify novel therapeutic targets to improve patient outcomes. We analyzed single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) and bulk transcriptome data from The Cancer Genome Atlas (TCGA). Pseudo-time trajectory and cell-cell communication analyses were performed with the Monocle and CellChat packages. The Wilcoxon test was used to evaluate the differential gene expression between wild-type (wt) and mutant (mut) groups. Prognostic models were developed using the Least Absolute Shrinkage and Selection Operator (LASSO) algorithm and Cox regression analyses to find the genes related to survival, with survival analysis conducted via Kaplan-Meier curves. Gene set enrichment analysis (GSEA) was employed to investigate pathway dysregulation, and immune cell infiltration was assessed using various immune scoring methodologies to explore the differences immune environment. The Tumor Immune Dysfunction and Exclusion (TIDE) database was utilized to predict the potential efficacy of immune checkpoint inhibitors. mRNA and protein expression levels of SPP1 were examined by RT-qPCR and Western blotting, while cell proliferation was assessed using the CCK8 assay. The mut group demonstrated significant alterations in cellular composition, characterized by increased endothelial cells and macrophages and decreased fibroblasts and CD8 + T cells, indicative of an immunosuppressive TME. Differential expression analysis revealed downregulation of immune pathways, including antigen processing and presentation, T cell receptor signaling, and B cell receptor signaling pathways in the mut group, along with enhanced metabolic activity in glycolysis and lipid metabolism. The prognostic model identified nine key genes associated with poor survival in HNSCC. The mut group exhibited poorer overall survival and a more immunosuppressive microenvironment compared to the wt group, which correlated with the outcomes observed in high-risk versus low-risk groups. High-risk patients also showed a diminished response to immunotherapy compared to low-risk patients. Additionally, SPP1 emerged as a critical gene associated with chemotherapy resistance and macrophage M2 polarization. This study demonstrates that 3p deletion significantly reshapes the TME, contributing to poor prognosis in HNSCC by fostering an immunosuppressive environment and enhancing chemoresistance. These findings highlight the potential for developing targeted therapies that address the genetic and immunological landscape of HNSCC.