Dexamethasone (Dex) is considered as the main steroid routinely used in the standard therapy of brain tumor-induced edema. Strong immunosuppressive effects of Dex on effector systems of the immune system affect the patients' antitumor immunity and may thereby worsen the prognosis. Siglecs and their interacting sialoglycans have been described as a novel glyco-immune checkpoint axis that promotes cancer immune evasion. Despite the aberrant glycosylation in cancer is described, mechanisms involved in regulation of immune checkpoints in gliomas are not fully understood. The aim of this study was to investigate the effect of Dex on the Siglec-sialic acid interplay and determine its significance in immune inversion in monocultured and co-cultured microglia and glioma cells. Both monocultured and co-cultured in transwell system embryonic stem cell-derived microglia (ESdM) and glioma GL261 cells were exposed to Dex. Cell viability, immune inversion markers, and interaction between sialic acid and Siglec-E were detected by flow cytometry. Cell invasion was analyzed by scratch-wound migration assay using inverted phase-contrast microscopy. Exposure to Dex led to significant changes in IL-1β, IL-10, Iba-1, and Siglec-E in co-cultured microglia compared to naïve or monocultured cells. These alterations were accompanied by increased α2.8-sialylation and Siglec-E fusion protein binding to co-cultured glioma cell membranes. This study suggests that the interplay between sialic acids and Siglecs is a sensitive immune checkpoint axis and may be crucial for Dex-induced dampening of antitumor immunity. The targeting of sialic acid-Siglec glyco-immune checkpoint can be a novel therapeutic method in glioma therapy.