Notch signalling pathway, particularly its ligand delta-ligand 3 (DLL3), is important in glioma, however, little is known about DLL3 regulation and prognostic effects. Immunohistochemistry on a cohort of 163 gliomas revealed DLL3 upregulation in IDH1 mutant gliomas, where it was associated with a favourable prognosis (HR[95% CI]: 0.28[0.09–0.87]; p = 0.021). We investigated the epigenetic regulation of DLL3, and identified individual CpG sites correlating with DLL3 mRNA expression, which were significant prognostic markers in LGG. In silico analysis revealed that infiltrating immune cells significantly correlated with DLL3 expression, methylation and somatic copy number alterations. The prognostic effects of DLL3 expression was significantly affected by infiltration of immune cells. RNA Sequencing of 83 LGGs and GO Term analysis of differentially expressed genes showed that low DLL3 expression was related to ciliogenesis, which was confirmed by TCGA LGG analysis. Thus, DLL3 may play an important role in the immune microenvironment and prognosis of LGGs.