Glycosylation is the most common posttranslational modification of proteins. Glycosyltransferase gene differential expression dictates the glycosylation model and is epigenetically regulating glioma progression and immunity. This study is aimed at identifying the glycosyltransferase gene signature to predict the prognosis and immune characteristics of glioma. The glycosyltransferase gene signature of glioma was identified in the TCGA database and validated in the CGGA database. Glioma patients were then divided into high- and low-risk groups based on risk scores to compare survival differences and predictive capacity. Subsequently, validation of glycosyltransferase gene signature merits by comparing with other signatures and utility in clinical judgment. The immune cell infiltration, immune pathways, and immune checkpoint expression level were also analyzed and compared in the high- and low-risk groups. Finally, the signature and its gene function were tested in our cohort and in vitro experiments. Eight glycosyltransferase genes were identified to establish the glycosyltransferase signature to predict the prognosis of glioma patients. The survival time was shorter in the high-risk group compared to the low-risk group based on glycosyltransferase signature and was confirmed in an independent external cohort. The glycosyltransferase signature displayed outstanding predictive capacity than other signatures in the TCGA and CGGA database cohorts. Furthermore, patients in the high-risk group were positively correlated with TAM infiltration, immune checkpoint expression level, and protumor immune pathways in TCGA cohorts. Validation of clinical tissue specimens revealed that the high-risk group was closely associated with infiltration of M2 TAMs. High-risk genes in the signature promote glioma proliferation, invasion, and macrophage recruitment in an in vitro validation of U87 and U251 cell lines. This carefully constructed that glycosyltransferase signature can predict the prognosis and immune profile of gliomas and help us evaluate subsequent macrophage-targeted therapies as well as other immune microenvironment modulation therapeutic strategies.Copyright © 2023 Yiwei Qi et al.