Although surgery and high‑dose radiotherapy have been the standard treatments for glioblastoma multiforme (GBM), these therapies are palliative, due to the high risk of local relapse. Emerging evidence has demonstrated that DNA double‑strand break (DSB) repair serves a critical role in resistance to radiotherapy. Previous studies have revealed that mangiferin possesses anti‑neoplastic effects on human lung adenocarcinoma and ovarian cancer. The present study aimed to investigate the role of mangiferin in radio‑sensitivity inhuman GBM. Through in vitro experiments, decreased proliferation and increased DNA damage were observed in cells pretreated with mangiferin following radiation. Further study of the repair pathway indicated that mangiferin inhibits the non‑homologous end‑joining (NHEJ) DSB repair pathway. Furthermore, studies on key proteins in the NHEJ DSB repair pathway revealed that mangiferin inhibited the phosphorylation of serine‑protein kinase ATM, TP53‑binding protein 1 and γ‑histone H2AX (γ‑H2AX). In addition, observations on the average percentages of γ‑H2AX‑positive cells and the average number of γ‑H2AX foci per cell suggested that treatment with mangiferin decreased the number of γ‑H2AX foci in GBM cells following radiation. However, mangiferin selectively inhibited DSB repair in GBM cells, and was not able to trigger DSB repair inhibition in normal neuronal Schwann cells. Through in vivo tumor‑bearing mouse experiments, a smaller tumor volume, decreased tumor weight and prolonged life span were observed in mice treated with mangiferin following radiation. Therefore, xenograft GBM models clearly demonstrated that treatment with mangiferin treatment may increase tumor sensitivity to radiotherapy. Taken together, as demonstrated by in vivo and in vitro data, mangiferin may be a potential novel therapeutic drug for improving the radiation sensitivity of glioblastoma.