Glioblastoma (GBM), as one of the most common and aggressive primary brain tumors, pose significant challenges in diagnosis and treatment, highlighting the urgent need for the identification of novel biomarkers, NFKBIE, which may play a critical role in tumor progression, immune modulation, and therapeutic response. We investigated the role of NFKBIE in GBM through both bioinformatics analysis and experimental validation. Initially, bioinformatics analysis based on public databases revealed significant differential expression of NFKBIE across various cancer types, particularly in GBM, where elevated expression was correlated with poor patient prognosis, including overall survival, disease-specific survival, and progression-free survival. Furthermore, genetic alterations in NFKBIE, such as copy number variations (CNVs) and tumor mutational burden (TMB), were significantly associated with tumor progression. In the experimental validation phase, we utilized small interfering RNA (siRNA) technology to silence NFKBIE expression in GBM cell lines, including U87 and T98G. Functional assays were then performed to assess the impact of NFKBIE knockdown. Cell proliferation assays, including CCK-8 and clonogenic assays, demonstrated that silencing NFKBIE significantly inhibited the proliferation of GBM cells. Migration and invasion assays, including wound healing and Transwell assays, further confirmed that NFKBIE knockdown markedly suppressed the migratory and invasive abilities of GBM cells. Additionally, apoptosis assays, such as TUNEL staining, revealed that NFKBIE silencing significantly promoted apoptosis in GBM cells. Western blot analysis confirmed the changes in the expression of key markers associated with proliferation, stemness, migration, invasion, and apoptosis. Furthermore, in a mouse xenograft model, NFKBIE knockdown significantly slowed tumor growth. Histological analysis of the excised tumors confirmed that reduced NFKBIE expression was closely associated with the inhibition of tumor growth. Notably, NFKBIE silencing also led to the downregulation of components of the Hedgehog signaling pathway, suggesting that NFKBIE may regulate tumor progression through modulation of this critical pathway. In conclusion, high NFKBIE expression is strongly associated with GBM progression and poor prognosis, and targeting its expression may offer a promising therapeutic strategy for GBM treatment.