MicroRNA-10b (miR-10b) is commonly elevated in glioblastoma (GBM), while not expressed in normal brain tissues. Targeted inhibition of miR-10b has pleiotropic effects on GBM derived cell lines, it reduces GBM growth in animal models, but does not affect normal neurons and astrocytes. This data raises the possibility of developing miR-10b-targeting GBM therapy. However, the mechanisms contributing to miR-10b-mediated glioma cell survival and proliferation are unexplored. We found that inhibition of miR-10b has distinct effects on specific glioma cell lines. In cells expressing high levels of tumor suppressor p21WAF1/Cip1, it represses E2F1-mediated transcription, leading to down-regulation of multiple E2F1 target genes encoding for S-phase specific proteins, epigenetic modulators, and miRNAs (e.g. miR-15/16), and thereby stalling progression through the S-phase of cell cycle. Subsequently, miR-15/16 activities are reduced and many of their direct targets are de-repressed, including ubiquitin ligase FBXW7 that destabilizes Cyclin E. Conversely, GBM cells expressing low p21 level, or after p21 knock-down, exhibit weaker or no E2F1 response to miR-10b inhibition. Comparative analysis of The Cancer Genome Atlas revealed a strong correlation between miR-10b and multiple E2F target genes in GBM and low-grade glioma. Taken together, these findings indicate that miR-10b regulates E2F1-mediated transcription in GBM, in a p21-dependent fashion.