Glioblastoma multiforme (GBM) is the most malignant primary brain tumor with dismal prognosis. This tumor is characterized by extensive heterogeneity, thus is difficult to treat and every established or new treatment faces significant hazard of resistance. Temozolomide (TMZ), an oral alkylating agent, is the first-line treatment for GBM, but resistance to TMZ is a major problem. Herewith, we investigated the combined effect of TMZ, difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, and radiation in GBM cell lines.We used the U87G, U251MG and T98G GBM cell lines. A linac 6MV accelerator (Varian Medical Systems) was used for cell irradiation. Viability and proliferation of the cells were examined with trypan blue exclusion assay, crystal violet and xCELLigence system. Cell cycle and activation of caspase-8 were evaluated with flow cytometry.The combination treatment resulted in a consistent higher suppression of proliferation in all cell lines treated and induced a significant higher cell cycle arrest in G2/M phase in U251MG and T98G cell lines. In U251MG cells caspase-8 was increased with each treatment alone, however the combination treatment had lower level of caspase-8 induction, suggesting a co-existence of another mechanism of cell death apart from apoptosis. In T98G cells the combination treatment increased the activation of caspase-8.Combination treatment with DFMO, TMZ and radiation significantly reduced cell viability in all cell lines tested. Given that both TMZ and DFMO can be administered orally and are related to minimal toxicities, this combination treatment may be a novel treatment strategy for GBM that deserves further investigation.