To study the method of isolating and culturing synovium-derived MSCs (SMSCs), and to investigate its multiple differentiation potential in vitro. Methods Three 2-month-old Changfeng hybrid swines weighing 8-10 kg (male and female) were used. SMSCs were harvested from the synovium of swine knee joints and cultured in vitro. When the SMSCs at passage 3 reached confluence, basic culture medium was removed, and the multi ple differentiationpotential of SMSCs was demonstrated in specific induction media (experimental group). The cells at passage 3 cultured with basic culture medium served as control group. After 21 days of chondrogenic differentiation, the cells underwent toluidine blue staining, immunohistochemistry staining and real-time fluorescence quantitative PCR detection. After 10 and 21 days of osteogenic differentiation, the cells underwent ALP staining and Al izarin red staining, respectively. After 21 days of adipogenic differentiation, the cells underwent Oil red O staining. Results SMSCs displayed long and thin or polygonal morphology 24 hours after culture. They prol iferated fast 48 hours after culture and presented large number of spindle-shaped cells with few globular cells 72 hours after culture. For the experimental group 21 days after chondrogenic induction, the cells were positive for toluidine blue staining with the formation of Aggrecan outside the cells; the immunohistochemistry staining revealed the expression of Col II; the real-time fluorescence quantitative PCR detection showed that the expressions of Col II A1, Aggrecan and SOX9 mRNA of the experimental group were greater than that of control group (P lt; 0.05). The cells were positive for ALP staining 10 days after osteogenic induction, and positive for Al izarin red staining 21 days after osteogenic induction, with the formation of calcium nodules. Oil red O staining displayed the formation of l i pid droplets inside the cells 21 days after adi pogenic induction. For the control group, the results of all the staining assays were negative except the ALP staining presenting with sl ight positive result. Conclusion SMSCs can be isolated from knee joint of swine and proliferate and differentiate into osteogenic, adi pogenic and chondrogenic cells in vitro. SMSCs may be a promising source of seed cells for tissue engineering.