Background: Tumor cells exhibit an increased requirement for iron to support their rapid proliferation. Deferoxamine (DFO), an iron chelator, has been reported to have anti-proliferative effects on cancer cells through induction of apoptosis and cell cycle arrest. X-rays also induce apoptosis and cell cycle arrest. However, limited information is available regarding the effect of iron depletion on radiotherapy. In this study, the radiosensitizing effect of DFO was investigated in human glioma U251 cells. Materials and Methods: U251 cells were pretreated with DFO before exposure to X-rays. The radiosensitizing effect of DFO on U251 cells was evaluated with a clonogenic formation assay. Apoptosis and autophagy were measured to explore the model of cell death during DFO radiosensitization. Intracellular calcium levels, cell cycle, and ROS levels were examined to study the mechanism of the cell death.  Results: We found that DFO enhanced X-ray-induced growth inhibition of U251 cells. Increased protective autophagy, occurring in U251 cells the first day of being treated with DFO and X-rays, rendered the radiosensitivity enhancement of DFO insignificant. However, the radiosensitizing effect was clear from the fourth day, which was attributed to the increase of apoptosis and decrease of protective autophagy. Further investigation revealed that the high level of apoptosis induced by DFO plus X-rays was dependent on the level of cytoplasmic calcium because the apoptosis was inhibited by [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], a cytoplasmic calcium chelator. Conclusion: Our results clearly demonstrate that DFO can enhance the radiosensitivity of U251 cells by increased calcium-dependent apoptosis.