Abstract Glioblastoma multiforme (GBM) are highly malignant primary brain tumors, and the Mesenchymal GBM subtype in particular, frequently exhibit regions of severe hypoxia and necrosis. As these features correlate with poor prognosis, we investigated miRNAs whose expression might regulate ischemic cell survival and growth. We found that miR-218 expression is significantly repressed in highly necrotic and chemoresistant Mesenchymal GBMs, compared to the Proneural subtype. Furthermore, restoring miR-218 expression in glioblastoma stem like cells (GSCs) significantly sensitizes orthotopic xenograft tumors to chemotherapeutic treatment, suggesting that low miR-218 confers GBM chemoresistance. Moreover, reduced miR-218 levels correlate with poor survival and rapid recurrence in human Mesenchymal GBM patients treated with radio- and chemotherapy (in contrast, no survival differences were observed in other GBM subtypes). Importantly, miR-218 targets multiple components of the receptor tyrosine kinase (RTK) signaling pathways, and reduced miR-218 expression increases their abundance and activity, thereby overcoming RTK inhibitory homeostatic mechanisms that normally modulate active signaling. We further demonstrate that HIFs, specifically HIF2α, function as downstream effectors of the low miR-218-RTK activation cascade in GSC cells. In strong support of our experimental findings, we observed a significant correlation between elevated HIF activity and low miR-218 expression in Mesenchymal GBM patient samples; in contrast, no correlation was observed in Proneural tumors. Altogether, our data reveal a novel functional association between miR-218 expression and HIF2α activity, and delineate RTK activation as the mechanistic link between the two in highly necrotic GBMs. Moreover, as tumor hypoxia is associated with chemoresistance and poor prognosis, our results uncover a new mechanism whereby miR-218 repression enhances HIF activity, and contributes to the chemoresistant phenotype predominantly seen in Mesenchymal GBM. Citation Format: Lijoy K. Mathew, Nicolas Skuli, Vera Mucaj, Samuel S. Lee, Sriram Venneti, Priti Lal, Justin D. Lathia, Jeremy N. Rich, Brian Keith, Andy J. Minn, M Celeste Simon. miR-218 opposes an important RTK–HIF signaling pathway in Glioblastoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B68. doi:10.1158/1538-7445.CHTME14-B68