Glioblastoma multiforme (GBM) is the most common, highly malignant primary tumor of the brain with poor prognosis. Even with the improved therapy regimen including temozolomide, the average survival rate is less than 2 years. Additional approaches to therapy targeting multiple aspects of glioma progression are in need. In the present work, we have tested the possibility that upregulation of the transcription factor interferon regulatory factor 3 (IRF3) can inhibit glioma invasiveness, proliferation and production of pro-inflammatory and pro-angiogenic factors in cultures of malignant glioma cell lines (U271, U87 and SNB-19). IRF3 is an essential transcription factor involved in TLR3/4-mediated signaling and generation of type I interferons. Although IRF3 has been suggested as a potential tumor suppressor gene, its role in glioma remains uninvestigated. In this study, we find that human glioma immune activation is potently elicited by a cytokine combination, IL-1/IFNγ (or poly IC), but not by bacterial lipopolysaccharide (LPS), similar to primary human astrocytes. GBM biopsy specimens show little detectable IRF3 immunoreactivity, and in vitro adenovirus-mediated IRF3 gene transfer in glioma cells modulates IL-1/IFNγ-induced cytokine and chemokine genes, resulting in upregulation of IFNβ and IP-10 (IRF3-stimulated genes) and downregulation of proinflammatory and angiogenic genes including IL-8, TNFα and VEGF (IRF3-represssed genes). Cytokines (IL-1β and TNFα) also induce the expression of miR-155 and miR-155*, the microRNAs crucial in immunity and inflammation-induced oncogenesis and this is dose-dependently suppressed by IRF3. Importantly, IRF3 also inhibits glioma proliferation, migration and invasion. Together, these data suggest that IRF3 can suppress glioma progression. Agents that promote IRF3 activation and expression (such as IRF3 gene transfer) could be explored as potential future therapy.