BACKGROUND: Glioma is a highly heterogeneous brain tumor with poor prognosis. This study aims to investigate the functional role of OTUB1 in glioma and its impact on TRAF4 stability, seeking potential therapeutic targets. METHODS: We mined single-cell sequencing data from 12 glioma patients to analyze the heterogeneity of 20,145 glioma cells. The expression of OTUB1 in glioma tissues and cell lines was assessed using Western blot and qPCR. Additionally, immunoprecipitation and ubiquitination assays were conducted to evaluate the effect of OTUB1 on TRAF4 and its role in regulating TRAF4 stability. In vitro assays were performed to assess the effects of OTUB1 on cell proliferation, migration, and clonogenicity, while in vivo experiments using xenograft models in nude mice validated the impact of OTUB1 on tumor growth. RESULTS: OTUB1 was found to be significantly overexpressed in glioma tissues, correlating with poor patient outcomes. Knockdown of OTUB1 markedly inhibited the proliferation and migration of LN229 and U87MG cells while increasing apoptosis. Immunoprecipitation studies revealed that OTUB1 stabilizes TRAF4 by inhibiting its ubiquitination, thereby promoting glioma cell proliferation and invasion. In vivo, tumors with OTUB1 knockdown demonstrated significantly reduced growth rates. CONCLUSION: OTUB1 plays a critical role in glioma progression and may serve as a novel therapeutic target. The development of inhibitors targeting OTUB1 could potentially improve outcomes for glioma patients.