Objective To explore the effect of synovial fluid of osteoarthritis (OA) on the differentiation of synovium-derived stem cells (SDSCs). Methods The SDSCs from the synovium of OA patients were identified and were induced to cartilage differentiation. The SDSCs were cultured with OA joint fluid (experimental group) or without OA joint fluid (control group). The diameter of the cartilage was measured. The mRNA and protein expressions of COL Ⅱ, Aggrecan and SOX9 were determined by RT-PCR and Western blotting analysis. Results The diameter of the cartilage in the experimental group was greater than that in the control 16 days after cultured with OA joint fluid (P<0.05). And the levels of COLⅡ, Aggrecan and SOX9 mRNA and protein in the experimental group was significantly higher than those in the control group (P<0.05). Conclusion The OA joint fluid can promote differentiation of SDSCs.