Clinical treatment of cartilage defects is challenging due to concomitant post-traumatic joint inflammation. This study was to demonstrate that the antioxidant ability of human adult synovium-derived stem cells (SDSCs) could be enhanced by ex vivo expansion on a decellularized stem cell matrix (DSCM). Microarray was used to evaluate oxidative, antioxidative, and chondrogenic status in SDSCs after expansion on the DSCM and induction in the chondrogenic medium. Hydrogen peroxide (H2O2) was added to create oxidative stress in either expanded SDSCs or chondrogenically induced premature pellets. The effect of H2O2 on SDSC proliferation was evaluated using flow cytometry. Chondrogenic differentiation of expanded SDSCs was evaluated using histology, immunostaining, biochemical analysis, and real-time polymerase chain reaction. Mitogen-activated protein kinase signaling pathways and p21 were compared in the DSCM and plastic-flask-expanded SDSCs with or without H2O2 treatment. We found that expansion on the DSCM upregulated antioxidative gene levels and chondrogenic potential in human SDSCs (hSDSCs), retarded the decrease in the cell number and the increase in apoptosis, and rendered SDSCs resistant to cell-cycle G1 arrest resulting from H2O2 treatment. Treatment with 0.05 mM H2O 2 during cell expansion yielded pellets with increased chondrogenic differentiation; treatment in premature SDSC pellets showed that the DSCM-expanded cells had a robust resistance to H2O 2-induced oxidative stress. Extracellular signal-regulated kinases 1 and 2 and p38 were positively involved in antioxidative and chondrogenic potential in SDSCs expanded on the DSCM in which p21 was downregulated. DSCM could be a promising cell expansion system to provide a large number of high-quality hSDSCs for cartilage regeneration in a harsh joint environment. © Copyright 2013, Mary Ann Liebert, Inc. 2013.