Bufalin, a cardiotonic steroid found in the venom of the Chinese toad Bufo gargarizan, has been shown to inhibit the growth of human cancers, such as colon and bladder. Here, we investigated the response of U251 and U87MG glioblastoma (GBM) cell lines to bufalin in vitro and found that bufalin impaired several biological processes. First, in both U251 and U87 MG, bufalin reduced cell proliferation and induced a G2/M cell cycle arrest (∼10% vs∼30%, untreated vs treated cells, respectively). Second, bufalin disrupted the mitochondrial membrane potential, leading to reduced oxygen consumption and ATP production. Third, homologous recombination (HR) efficiency was reduced by∼40% in both cell lines in the presence of bufalin. At the molecular level, bufalin led to decreased RAD51 protein, a central player in HR, and increased γ-H2AX, a marker for the presence of DNA double strand breaks. Finally, bufalin was additive with radiation in the treatment of GBM cells in vitro. Cell death increased significantly under combination treatment compared to radiation treatment alone. Our findings indicated that bufalin led to reduced mitochondrial and DNA repair function and therefore, might be a promising therapeutic drug to increase the sensitivity of GBM cells to radiotherapy.