The repair of large bone defects has always been a challenge, especially with respect to regeneration capacity and autogenous bone availability. To address this problem, we fabricated a 3D-printed polylactic acid (PLA) and hydroxyapatite (HA) scaffold (3D-printed PLA-HA, providing scaffold) loaded with enhanced bone marrow (eBM, providing seed cells) combined with induced membrane (IM, providing grow factors) to repair large radial defects in rabbits. in vitro assays, we demonstrated that 3D-printed PLA-HA had excellent biocompatibility, as shown by co-culturing with mesenchymal stem cells (MSCs); eBM-derived MSCs exhibited considerable differentiation potential, as shown in trilineage differentiation assays. To investigate bone formation efficacy in vivo, the rabbit radial long bone defect model was established. In the first stage, polymethylmethacrylate (PMMA) was inserted into the bone defect to stimulate the formation of IM; in the second stage, iliac crest bone graft (ICBG) with IM, PLA-HA alone with the removal of IM, PLA-HA with IM, and PLA-HA in conjunction with IM and eBM were sequentially applied to repair the long bone defect. At 8, 12, and 16 weeks, X-ray plain radiography, microcomputed tomography, and histological analysis were performed to evaluate the efficacy of bone repair and bone regeneration in each group. We found that IM combined with PLA-HA and eBM prominently enhanced bone repair and reconstruction, equivalent to that of IM/ICBG. Taken together, the data suggest that PLA-HA loaded with eBM combined with IM can be an alternative to IM with bone autografts for the treatment of large bone defects.