This study aimed to investigate the efficacy of three-dimensional scaffolds of silk fibroin/chitosan/nano-hydroxyapatite (SF/CS/nHA) and bone marrow derived mesenchymal stem cells (BMSCs) on the repair of long segmental bone defects in rabbits. BMSCs were cultured with SF/CS/nHA in vitro, and cell proliferation, alkaline phosphatase activity and Ca2+ content were examined. A 15 mm segmental defect in the radius was generated in 12 New Zealand White rabbits, which were divided randomly into three groups (n = 4): experimental group with SF/CS/nHA scaffold of induced BMSCs; control group with SF/CS/nHA scaffold; and blank group without any materials. Postoperatively at 12 weeks, osteogenesis effect and the degradation and absorption of SF/CS/nHA were evaluated by X-ray, hematoxylin eosin staining, and scanning electron microscopy. In vitro, SF/CS/nHA scaffolds exhibited good biocompatibility and no toxicity. SF/CS/nHA promoted adhesion, growth, and calcium nodule formation of BMSCs compared to control (P < 0.05). In vivo, we observed gradual new bone formation and bone defect gradually recovered at 12 weeks in experimental and control group, but more new bone was formed in experimental group (P < 0.05). In blank group, limited bone formation was observed and bone defect was obvious. In conclusion, SF/CS/nHA scaffolds loaded with BMSCs achieve high efficacy to repair segmental defect in the radius.