Deoxyribonuclease 2 (DNASE2) is associated with tumor proliferation and apoptosis, innate immune signaling, chronic inflammation, and systemic autoinflammatory diseases. However, the role and mechanism of DNASE2's action in gliomas remain unclear. In this study, the difference analysis showed that after supplementing normal tissue samples from the Genotype-Tissue Expression (GTEx) dataset, DNASE2 mRNA levels in 30 tumors from The Cancer Genome Atlas (TCGA) showed significant differences, correlated with a poor prognosis in patients with glioblastoma multiforme (GBM). DNASE2 down-regulation also reduced GBM cell proliferation, migration, and invasion. DNASE2 affected the immune activity of GBM cells. Furthermore, we found that interleukin-17, Toll-like receptor, North signaling pathway, secreted phosphoprotein 1 (SPP1), and S100 calcium-binding protein A8/9 (S100A8/9) genes may be crucial to regulate GBM immunity via DNASE2. In conclusion, DNASE2 expression is elevated in patients with GBM and influences the development of GBM, possibly through various immune-related pathways. Therefore, DNASE2 may serve as a potential prognostic biomarker for GBM. The overall workflow of this study is shown in Figure 1A.