Background <p>E2F transcription factors are key regulators of the cell cycle and play an important role in tumorigenesis. N6-methyladenosine (m⁶A), the most prevalent internal RNA modification in eukaryotic cells, is also crucial for cancer progression. However, the interplay between E2F family members and m⁶A modification in glioblastoma multiforme (GBM) remains poorly understood.</p> Methods <p>We applied weighted gene co-expression network analysis (WGCNA) to identify m⁶A-related gene modules co-expressed with <i>E2F7</i>, a gene that is significantly associated with adverse prognosis and tumor stemness. Through intersection analysis, eukaryotic translation initiation factor 3 subunit B (<i>EIF3B</i>) emerged as a hub gene, showing a strong positive correlation with <i>E2F7</i> in GBM datasets. Functional validation was performed in GBM cells to assess the regulatory relationship between EIF3B and E2F7, including examination of mRNA and protein expression, mRNA stability, and m⁶A modification levels. GBM stemness was evaluated by tumor sphere formation assays and detection of stemness-associated markers.</p> Results <p>Functional validation revealed that EIF3B knockdown reduced E2F7 mRNA and protein expression, promoted <i>E2F7</i> mRNA degradation, and decreased its m⁶A modification, suggesting an association between EIF3B and m⁶A-related regulation of E2F7 stability. Furthermore, EIF3B knockdown markedly suppressed GBM stemness, as demonstrated by impaired tumor sphere formation and downregulation of stemness-associated markers. Clinically, EIF3B expression increased with glioma grade and correlated with poor prognosis.</p> Conclusions <p>Collectively, these findings suggest that the EIF3B-E2F7 axis is associated with m⁶A-related regulation in GBM and may warrant further investigation as a candidate for therapeutic targeting.</p>