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Background/Objectives: Glioblastoma (GBM) is the most aggressive type of brain tumor in adults. Currently, the only treatments available are surgery, radiotherapy, and chemotherapy based on temozolomide (TMZ); however, the prognosis is dismal. Several natural substances are under investigation for cancer treatment. 8α-O-(3,4-dihydroxy-2-methylenebutanoyloxy) dehydromelitensine (Isocnicin) is a natural compound derived from Centaurea species and was found to exhibit cytostatic/cytotoxic effect against different cell lines. In this study, we investigated the anti-glioma effects of isocnicin in U87 and T98 glioblastoma cell lines, as well as the effects of combined treatment with radiotherapy. Methods: Cell viability was evaluated with the trypan blue exclusion assay, cell cycle distribution was examined using flow cytometry, and the effects of the combination treatment were analyzed with CompuSyn software(1.0). Results: The result showed that isocnicin significantly reduced cell viability in U87 and T98 cell lines in a dose-dependent manner and IC50 values were calculated. Administration of isocnicin alone induced both S and G2/M cell cycle arrest in U87 and T98 cells in a dose-dependent manner. Moreover, when cells were treated with increasing concentrations of isocnicin, followed by 2 or 4 Gy of radiation, the percentage distribution of the cells in the G2/M phase was increased considerably in both U87 and T98 cell lines. Conclusions: Here, we show for the first time that co-treatment of isocnicin with radiation exerts a synergistic antiproliferative effect in glioblastoma cell lines. Natural compounds are promising for glioblastoma treatment. Further studies will be necessary to unravel isocnicin's mechanism of action and its synergistic effect with radiation on glioblastoma treatment. [ABSTRACT FROM AUTHOR]