Glioblastoma is a highly malignant and typically fatal tumor of the central nervous system. The tumor is characterized by marked cellular and molecular heterogeneity, including a subpopulation of brain tumor initiating cells (BTICs) that are highly resistant to radiation and chemotherapy. We previously reported that the RNA-binding protein HuR is: (1) overexpressed in glioblastoma, (2) necessary for tumor growth in vivo, and (3) a positive regulator of tumor-promoting genes in glioblastoma. These findings provide strong evidence that HuR might be a viable therapeutic target in glioblastoma. In this report, we investigated the effects of MS-444, a small molecule inhibitor of HuR, in xenograft-derived human glioblastoma cells and BTICs. We found that MS-444 treatment of glioblastoma cells resulted in loss of viability and induction of apoptosis, with evidence implicating death receptor 5. BTICs were particularly sensitive to MS-444. At sub-lethal doses, MS-444 attenuated invasion of glioblastoma cells and BTICs in a transwell model. At the molecular level, MS-444 treatment led to an attenuation of mRNAs in different tumor promoting pathways including angiogenesis, immune evasion and suppression of apoptosis. Although cytoplasmic HuR was reduced with MS-444 treatment, the attenuation of mRNAs could not be explained by RNA destabilization. In summary, this report provides proof of concept that small molecule inhibition of HuR could be a viable approach for treatment of glioblastoma.