Objective To investigate the effect of various concentration of platelet-rich plasma (PRP) on osteogenic differentiation of rabbit skeletal muscle-derived stem cells (SMSCs) cultured in vitro. Methods Blood drawn from the central ear arteries of 9 one-year-old New Zealand white rabbits weighing 2.5-3.0 kg (male and female) was used to prepare PRP (Landesberg method). Full blood count and platelet count in PRP were tested. Soleus muscle of right hindl imb in rabbit was obtained and used to culture SMSCs in vitro. The cells at passage 3 were randomly divided into different groups: the experimental groups in which the cells were treated by conditioned culture media with various concentrations of autologousPRP (6.25%, 12.50%, 25.00%, 50.00%), and the control group in which the cells were treated with the media without PRP. At different time points after intervention, osteogenetic activity of the cells was detected by ALP staining observation, ALP activity detection was conducted, al izarin red staining for calcium nodules and immunofluorescence staining for osteocalcin were performed, and core binding factor α1 (Cbfα1) of osteogenic gene expression was tested by RT-PCR. Results The full blood PRP count and the platelet count in PRP was (3.06 ± 0.46) × 105/μL and (18.08 ± 2.10) × 105/μL, respectively. ALP staining: the cells in all the experimental groups were positive for the staining with many black sediment particles in cytoplasm; the cells in the control group were negative staining. ALP activity: all the experimental groups were higher than the control group (P lt; 0.05), the experimental group at 12.50% was superior to other experimental groups at each time point (P lt; 0.05). Al izarin red staining: at 14 days after culture, orange-red calcium nodules were evident in all the experimental groups; no orange-red calcium nodules were observed in the control group with a mineral ization rate of zero; there were significant difference between the experimental groups and the control group in terms of mineral ization rate (P lt; 0.05), the experimental group at 12.50% had a higher mineral ization rate than other experimental groups (P lt; 0.05). Immunofluorescence staining for osteocalcin: at 7 days after culture, the experimental groups were positive for the staining with yellow fluorescence in cytoplasm, and the result of the control group was negative. RT-PCR detection: no obvious changes of the gene expression were noted at 4, 12, and 24 hoursafter culture in the control group; the gene expression in all the experimental groups was significant superior to that of control group, especially at 12 hours, and the expression in the experimental group at 12.50% was the highest. Conclusion PRP can obviously promote the osteogenic differentiation of SMSCs cultured in vitro in a concentration-dependent manner, and the 12.50% is proved to be the ideal concentration.