MicroRNA is an important regulator of glioblastoma. This study aims at validating microRNA-221 (miR-221) as a biomarker for glioblastoma, and understanding how miR-221 regulates glioblastoma progression. Using clinical samples, miR-221 expression was analyzed by quantitative reverse-transcriptase PCR (qPCR). SHG-44 cells were treated with anti-miR-221 or U87MG-derived exosomes followed by monitoring changes in cell viability, migration and temozolomide (TMZ) resistance. Bioinformatics approach was used to identify targets of miR-221. The interaction between miR-221 and its target, DNM3 gene, was studied with dual-luciferase reporter assay, Spearman’s correlation analysis, and western blotting. To verify that RELA regulates miR-221 expression, RELA-expressing vector or shRNA was introduced into SHG-44 cells and its effect on miR-221 expression was monitored. Both tissue-level and exosomal miR-221 expression increased with glioma grades. In SHG-44 cells, downregulating miR-221 expression inhibited cell proliferation, migration, and TMZ resistance, whereas incubation with U87MG-derived exosomes exerted tumor-promoting effects. DNM3 gene is a target of miR-221. RELA induced miR-221 expression. In glioma, elevated miR-221 expression is a biomarker for glioma. DNM3 is a target of miR-221 and RELA regulates miR-221 expression. The RELA/miR-221 axis is a target for glioma diagnosis and therapy.