Runt-Related Transcription Factor 1 (RUNX1) is highly expressed in the Mesenchymal (Mes) subtype of glioblastoma (GBM). However, the specific molecular mechanism of RUNX1 in Mes GBM remains largely elusive. In this study, cell and tumor tissue typing were performed by RNA-sequencing. Co-immunoprecipitation (co-IP) and immunofluorescence (IF) were employed to identify members of the RUNX1 transcriptional protein complex. Bioinformatics analysis, chromatin immunoprecipitation (ChIP), and luciferase reporter experiments were utilized to verify target genes. Analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) verified the expression levels and prognoses associated with RUNX1/p-SMAD3/SUV39H1 target genes. In vivo patient-derived xenograft (PDX) studies and in vitro functional studies verified the impact of RUNX1 on the occurrence and development of GBM. The results showed that RUNX1 was upregulated in Mes GBM cell lines, tissues and patients and promoted proliferation and invasion in GBM in a TGF beta pathway-dependent manner in vivo and in vitro. We found and verified that BCL3 and MGP are transcriptionally activated by p-SMAD3 /RUNX1, while MXI1 is transcriptionally suppressed by the RUNX1/SUV39H1-H3K9me3 axis. This finding offers a theoretical rationale for using molecular markers and choosing therapeutic targets for the Mes type of GBM.