Osteosarcoma is a kind of high-risk sarcoma of the skeleton typically observed in people under 25 years old. Currently, radiotherapy is widely applied in cancer treatment. However, osteosarcoma is radioresistant and accordingly new, more effective radiosensitizers are needed. miRNAs have been reported to play an important role in osteosarcoma radiosensitivity. We examined the modulating effect of miR‑328‑3p in vivo and in vitro. miR‑328‑3p was downregulated in HOS‑2R cells. The overexpression of miR‑328‑3p enhanced the radiosensitivity of osteosarcoma cells. miR‑328‑3p inhibited proliferation and promoted apoptosis in osteosarcoma cells under radiation conditions. In cells overexpressing miR‑328‑3p, H2AX expression was downregulated. We found that miR‑328‑3p targets H2AX and inhibits its expression. It was concluded, that miR‑328‑3p enhances the radiosensitization of osteosarcoma following X-ray irradiation, and determined that it directly targets H2AX to regulate radiosensitization.