Bone marrow-derived mesenchymal stem cell (BMSC) transplantation is considered a promising therapeutic approach for bone defect repair. However, during the transplantation procedure, the functions and viability of BMSCs may be impaired due to extended durations of in vitro culture, aging, and disease conditions of patients. Inspired by spontaneous intercellular mitochondria transfer that naturally occurs within injured tissues to rescue cellular or tissue function, we investigated whether artificial mitochondria transfer into pre-transplant BMSCs in vitro could improve cellular function and enhance their therapeutic effects on bone defect repair in situ. Mitochondria were isolated from donor BMSCs and transferred into recipient BMSCs of the same batch and passage. Subsequently, changes in proliferative capacity and cell senescence were evaluated by live cell imaging, Cell Counting Kit-8 assay, cell cycle analysis, Ki67 staining, qPCR and Western blot analysis of c-Myc expression, and β-galactosidase staining. Migration ability was evaluated by the transwell migration assay, wound scratch healing, and cell motility tests. Alkaline phosphatase (ALP) staining, Alizarin Red staining, and combined with qPCR and Western blot analyses of Runx2 and BMP2 were performed to elucidate the effects of mitochondria transfer on the osteogenic potential of BMSCs in vitro. After that, in vivo experiments were performed by transplanting mitochondria-recipient BMSCs into a rat cranial critical-size bone defect model. Micro CT scanning and histological analysis were conducted at 4 and 8 weeks after transplantation to evaluate osteogenesis in situ. Finally, in order to establish the correlation between cellular behavioral changes and aerobic metabolism, OXPHOS (oxidative phosphorylation) and ATP production were assessed and inhibition of aerobic respiration by oligomycin was performed. Mitochondria-recipient BMSCs exhibited significantly enhanced proliferation and migration, and increased osteogenesis upon osteogenic induction. The in vivo results showed more new bone formation after transplantation of mitochondria-recipient BMSCs in situ. Increased OXPHOS activity and ATP production were observed, which upon inhibition by oligomycin attenuated the enhancement of proliferation, migration, and osteogenic differentiation induced by mitochondria transfer. Mitochondria transfer is a feasible technique to enhance BMSC function in vitro and promote bone defect repair in situ through the upregulation of aerobic metabolism. The results indicated that mitochondria transfer may be a novel promising technique for optimizing stem cell therapeutic function.