Rapid tumor growth, widespread brain-invasion, and therapeutic resistance primarily contribute to an inevitable tumor recurrence and dismal patient outcome in glioblastoma. Although GBM stem cells (GSCs) are shown to play key roles in these processes, the molecular pathways driving the GSC phenotype (GBM-stemness) remain poorly defined. A global miRNA expression analysis was performed using FFPE GBM tumor samples, and we found that low expression of miR-146a correlated with poor clinical outcome and tumor recurrence. Based on this finding, the hypothesis is that miR-146a functions as a tumor suppressor and inhibits stemness in GBM. Expression analysis was performed using FFPE GBM tumor samples (n = 268) followed by univariable (UVA) and multivariable (MVA) analyses. Functional studies were conducted to define the role of miR-146a-POU3F2/SMARCA5 pathway in GBM tumorigenesis and therapeutic response. MicroRNA-146a was one of the top miRNAs that correlated with better prognosis in GBM patients by UVA analyses (p = 9.21E-05), which was independent of MGMT promoter methylation by MVA analyses (p<0.001). miR-146a expression was significantly downregulated in recurrent GBM tumors compared with the paired primary GBM tumors (p = 0.003). Overexpression of miR-146a significantly inhibited tumor cell growth and sensitized patient-derived primary GBM cells to temozolomide (TMZ) treatment in vitro, and showed statistically significant smaller tumor size (p<0.01) and prolonged survival (p = 0.001) in vivo. In addition, miR-146a was downregulated in glioma cancer stem cells, and overexpression of miR-146a significantly reduced stemness. Mechanistically, miR-146a directly silenced POU3F2 and SMARCA5, two transcription factors that mutually regulated each other, significantly compromising GBM-stemness and increasing TMZ response. Moreover, overexpression of miR-146a significantly inhibited NF-κB pathways through targeting POU3F2 and SMARCA5 in GBM. Collectively, our data show that miR-146a-POU3F2/SMARCA5 pathway plays a critical role in suppressing GBM-stemness and increasing TMZ-response, suggesting that POU3F2 and SMARCA5 may serve as novel therapeutic targets in GBM. Fundings: R01CA188228 (to A.C., R.B., K.L., and J.S.B-S.), and the T&P Bohnenn Fund for Neuro-Oncology Research (to P.A.R.). R01CA169368, R01CA11522358, R01CA1145128, R01CA108633, 1RC2CA148190, and U10CA180850-01; A Brain Tumor Funders Collaborative Grant; Ohio State University Comprehensive Cancer Center Award (all to A.C.).