microRNAs (miRNAs) play an important role in the pathology of glioblastoma (GBM), which is the most malignant and most common primary malignant brain tumor. miRNAs can target multiple genes simultaneously and are considered as potential therapeutic agents or targets. This study aimed to determine the role of miR-3174 in the pathobiology of GBM using both in vitro and in vivo approaches. This is the first study deciphering the role of miR-3174 in GBM. We studied the expression of miR-3174 and found it to be downregulated in a panel of GBM cell lines, GSCs and tissues relative to astrocytes and normal brain tissue. This finding led us to hypothesize that miR-3174 has a tumor-suppressive role in GBM. Exogenous expression of miR-3174 inhibited GBM cell growth and invasion, and hampered the neurosphere formation ability of GSCs. miR-3174 downregulated the expression of multiple tumor-promoting genes including CD44, MDM2, RHOA, PLAU and CDK6. Further, overexpression of miR-3174 reduced tumor volume in nude mice with intracranial xenografts. Immuno-histochemical study of brain sections with intracranial tumor xenografts revealed the pro-apoptotic and anti-proliferative activity of miR-3174. In conclusion, we demonstrated that miR-3174 has a tumor-suppressive role in GBM and could be exploited for therapeutic purposes.