Due to the limitation of clinical autologous bone supply and other issues, the development of bone regeneration materials is still a hot topic. Natural tissue-derived bone repair materials have good biocompatibility and degradability, but their structure and properties are likely to be adversely affected during terminal sterilization. In this study, a composite scaffold consisting of the acellular extracellular matrix and dicalcium phosphate (ECM/DCP) was fabricated and terminally sterilized by γ-ray irradiation. In addition, the ECM/DCP scaffold was saturated with water and was also sterilized by γ-ray irradiation (RX-ECM/DCP). Results showed that the triple helix structure of collagen was better maintained in RX-ECM/DCP than in ECM/DCP. The thermal stability of RX-DCP/ECM was much better than that of ECP/ECM. The in vitro and in vivo performances of both types of scaffolds were also evaluated. The RX-ECM/DCP scaffold exhibited better in vitro bioactivity than that of the ECM/DCP scaffold as evidenced by more mineral formation in the simulated body fluid. In addition, RX-ECM/DCP also induced more effective bone regeneration than the ECM/DCP scaffold did in a rat calvarial defect model. Results sufficiently demonstrated that the addition of water to the scaffold could protect the structure of the ECM/DCP scaffold from being damaged by γ-ray irradiation during the terminal sterilization process. In summary, this study demonstrated a means to protect the ECM structure, which in turn led to the improvement of bone regenerative properties of the materials during γ-ray irradiation of ECM-based bone repair materials.