Glioma-initiating cells (GICs) have stem-like cell properties thought to be sufficient for recurrence, progression and drug resistance in glioblastomas. In the present study, we defined miR-340 as a differentially expressed microRNA (miRNA) in human GICs that inhibit GIC-mediated tumorigenesis. Further, we defined tissue plasminogen activator (PLAT) as a critical direct target of miR-340 for inhibition. Among miRNAs screened we found that miR-340 expression was decreased in all human GICs and in human glioblastoma tissues, compared to human neural stem cells and normal brain tissues. miR-340 overexpression in GICs suppressed their proliferative, invasive and migratory properties in vitro, triggering cell senescence in vitro and inhibiting GIC-induced tumorigenesis in mouse brains. shRNA-mediated silencing of PLAT in GICs phenocopied the effects of miR-340 overexpression in vitro and in vivo, suggesting a potential role for tissue factor in stem-like cell function. Taken together, our results identified miR-340 as a tumor suppressor that functions in GIC to enforce PLAT blockade and ablate their stem-like functions.