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Abstract Background Mesenchymal stem cells (MSCs) have great potential for the repair and regeneration of bone fracture, but their optimal origins remain controversial. Methods Bone marrow-MSCs (BM-MSCs) and bone-bone marrow-MSCs (B-BM-MSCs) were isolated from 12 SD rats, and the morphology, MSC-associated markers, and proliferative capacity of these cells were compared using an inverted microscope, flow cytometry, and CCK-8 assays, respectively. After 14 days of osteoblastic induction, osteoblast phenotypes were detected by ALP and calcium nodule staining, and the expression of BMP-2 and TGF-β1 was observed by western blotting. Then, the rat tibia fracture model was established with 3 groups (n = 6 per group), the control, BM-MSC, and B-BM-MSC groups. Computed tomography (CT) imaging was performed to evaluate fracture healing at weeks 2, 4, and 6. Finally, the fractured bones were removed at weeks 4 and 6, and HE staining was performed to evaluate fracture healing. Results Although the 2 types of MSCs shared the same cellular morphology and MSC-associated markers, B-BM-MSCs had a higher proliferative rate than BM-MSCs from day 9 to day 12 (p < 0.05), and the expression levels of ALP and calcium were obviously higher in B-BM-MSCs than in BM-MSCs after osteogenic induction (p < 0.01 and p < 0.001, respectively). Western blot results showed that the expression levels of BMP-2 and TGF-β1 in B-BM-MSCs were higher than in BM-MSCs before and after osteogenic induction (p < 0.01). In the animal experiments, CT imaging and gross observation showed that B-BM-MSCs had a greater capacity than BM-MSCs to promote fracture healing, as the Lane-Sandhu scores of B-BM-MSCs at weeks 4 and 6 after operation (3.00 ± 0.81 and 9.67 ± 0.94, respectively) were higher than those of BM-MSCs (1.33 ± 0.47 and 6.67 ± 1.25, respectively; both p < 0.05). The HE staining results further supported this conclusion. Conclusions Taken together, our study results proved that MSCs obtained by co-culturing the bone and bone marrow from SD rats had better proliferative, osteogenic differentiation, and fracture healing capacities than BM-MSCs, perhaps suggesting a novel way to obtain MSCs for bone tissue repair.