Background: Hexokinase 3 (HK3) is one of the key enzymes involved in glucose phosphorylation (the first step in most glucose metabolic pathways). Many studies have demonstrated the vital role of dysregulation of HK3 in several tumors. However, there is a need for in-depth characterization of the role of HK3 in glioblastoma multiforme (GBM).Methods: All data were sourced from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA). Kaplan-Meier analysis and univariate regression were applied for survival analysis. Gene set enrichment analysis (GSEA) was used for enrichment analysis. Tumor Immune Single Cell Hub (TISCH) database was applied for single-cell analysis. Tumor Immune Dysfunction and Exclusion (TIDE) analysis was applied to evaluate the immune response.Results: HK3 expression was upregulated in GBM and correlated with poor prognosis. The high HK3 expression group was primarily enriched in adaptive immune response, chemokine signaling pathway, and cytokine-cytokine receptor interaction. The high HK3 expression group showed significantly greater enrichment of the majority of immune cells and immune-related pathways. HK3 showed significant correlation with most immune cells, especially macrophages (p < .001, R = .81). TISCH analysis showed that HK3 was predominantly expressed in macrophages in most cancers. HK3 showed significant correlation with most immune-related genes, such as PD-1 (p < .001, R = .41), PDL-1 (p < .001, R = .27), and CTLA-4 (p < .001, R = .29). TIDE analysis revealed that the low HK3 expression group has a lower TIDE score and may benefit from immunotherapy. Drug sensitivity analysis showed that patients with high HK3 expression frequently showed drug resistance.Conclusion: HK3 was associated with poor prognosis and may serve as a biomarker of macrophages in GBM. HK3 was also associated with immune response and drug resistance. Our findings may provide novel insights for GBM immunotherapy.