Mesenchymal stem cell-based therapy is a highly attractive strategy that promotes bone tissue regeneration. The aim of the present study was to evaluate the combination effect of muscle-derived mesenchymal stem cells (M-MSCs) and platelet-rich plasma (PRP) on bone repair capacity in rabbits with large humeral bone defect. Precise cylindrical bone defects of 10 mm diameter and 5 mm depth were established in rabbit humeral bones, which were unable to be repaired under natural conditions. The rabbits received treatment with M-MSCs/PRP gel, M-MSCs gel, or PRP gel, or no treatment. The bone tissue regeneration was evaluated at day 0-90 after surgery by HE morphological staining, Lane-Sandhu histopathological scoring, tetracycline detection, Gomori staining and micro-computed tomography. Beyond that, Transwell assay, CCK8 assay, Western blot analysis and ALP activity detection were performed in M-MSCs in vitro with or without PRP application to detect the molecular effects of PRP on M-MSCs. We found that the repair effect of M-MSCs group or PRP group was limited and the bone defects were not completely closed at post-operation 90 d. In contrast, M-MSCs/PRP group received obvious filling in the bone defects with a Lane-Sandhu evaluation score of 9. Tetracycline-labeled new bone area in M-MSCs/PRP group and new mineralized bone area were significantly larger than that in other groups. Micro-computed tomography result of M-MSCs/PRP group displayed complete recovery of humeral bone at post-operation 90 d. Further in vitro experiment revealed that PRP significantly induced migration, enhanced the growth, and promoted the expression of Cbfa-1 and Coll I in M-MSCs. In conclusion, PRP application significantly enhanced the regeneration capacity of M-MSCs in large bone defect via promoting the migration and proliferation of M-MSCs, and also inducing the osteogenic differentiation.