Glioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma (GBM), which is the most malignant form of glioma. The implications and underlying mechanisms of protein glycosylation in GSC phenotypes and GBM malignancy are not fully understood. In this study, we aimed to investigate the implication of protein glycosylation and the corresponding candidate genes on the stem cell properties of GSCs and poor clinical outcomes in GBM, using single-cell RNA sequencing and bulk RNA sequencing datasets of clinical GBM specimens deposited in the Gene Expression Omnibus database, in addition to The Cancer Genome Atlas and the Chinese Glioma Genome Atlas databases of patients with glioma. We demonstrated that N-linked glycosylation was significantly associated with GSC properties and the prognosis of GBM by conducting integrated bioinformatics analyses of clinical specimens. N-linked glycosylation was associated with the glioma grade, molecular biomarkers, and molecular subtypes. The expression levels of the asparagine-linked glycosylation (ALG) enzyme family, which is essential for the early steps in the biosynthesis of N-glycans, were prominently associated with GSC properties and poor survival in patients with GBM with high stem-cell properties. Finally, the oxidative phosphorylation (OXPHOS) pathway was primarily enriched in GSCs with a high expression of the ALG enzyme family. Collectively, these findings uncover a pivotal role for N-linked glycosylation in the regulation of GSC phenotypes and GBM malignancy through, possibly in part, the ALG-OXPHOS axis, thereby revealing a potential target for GSC-directed therapy.