The rehabilitation of bone defects after radiotherapy requires the development of osteoinductive bone substitutes. MicroRNA could be used as an osteogenic factor to fabricate functional materials for bone regeneration. In this study, we used miR-34a to enhance bone regeneration after irradiation. We lyophilized lipofectamine-agomiR-34a lipoplexes on hydroxyapatite (HA) to develop miR-34a-functionalized hydroxyapatite (HA-agomiR-34a). The morphology was observed by scanning electron microscope and atomic force microscope. Fluorescence microscopy confirmed the retention of agomiR-34a on the surface of HA. HA-agomiR-34a showed high transfection efficiency and good biocompatibility. HA-agomiR-34a enhanced the osteoblastic differentiation of radiation-impaired bone marrow stromal cells (BMSCs). Implantation of HA-agomiR-34a promoted bone regeneration in irradiated bone defects. HA-agomiR-34a may be a novel and safe bone substitute to promote the reconstruction of bone defects after radiotherapy.