Dolichyl-diphosphooligosaccharide protein glycosyltransferase (DDOST) plays a pivotal role in the glycosylation of asparagine residues on nascent polypeptides. However, the biological role of DDOST in glioma remains unclear. The mRNA expression of DDOST in glioma was identified using TCGA, CGGA, GEO and Rembrandt datasets. Immunohistochemistry assay was conducted to examine the protein level of DDOST. Cox regression analysis, nomograms and calibration plots were used to evaluate the prognostic value of DDOST. The association between DDOST and immune cell infiltration was evaluated using CIBERSORT algorithm. Additionally, DNA methylation and ceRNA regulatory network of DDOST expression were investigated using the LinkedOmics and ENCORI databases. The authors found that DDOST was substantially expressed at the mRNA and protein levels. Functional enrichment analysis revealed close associations between DDOST and immune-related pathways, as well as immune cell infiltration. In addition, DDOST exhibited synergistic effects with tumour mutational burden (TMB) and other immune checkpoints. For expression regulation mechanisms, DDOST had low DNA methylation levels in high-grade gliomas and may be involved in multiple ceRNA networks in glioma. Thus, DDOST may serve as an unfavourable biomarker for gliomas. DNA methylation and ceRNA regulatory networks of DDOST expression were identified for the first time in this multi-omics study.