Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are considered an attractive cell source for tissue regeneration. However, environmental oxidative stress can trigger premature senescence in MSCs and thus compromises their regenerative potential. Extracellular matrix (ECM) derived from MSCs has been shown to facilitate cell proliferation and multi-lineage differentiation. This investigation evaluated the effect of cell-deposited decellularized ECM (DECM) on oxidative stress-induced premature senescence in UC-MSCs. Sublethal dosages of H O , ranging from 50 μm to 200 μm, were used to induce senescence in MSCs. We found that DECM protected UC-MSCs from oxidative stress-induced premature senescence. When treated with H O at the same concentration, cell proliferation of DECM-cultured UC-MSCs was twofold higher than those on standard tissue culture polystyrene (TCPS). After exposure to 100 μm H O , fewer senescence-associated β-galactosidase-positive cells were observed on DECM than those on TCPS (17.6  ±  4.0% vs. 60.4  ±  6.2%). UC-MSCs cultured on DECM also showed significantly lower levels of senescence-related regulators, such as p16 and p21. Most importantly, DECM preserved the osteogenic differentiation potential of UC-MSCs with premature senescence. The underlying molecular mechanisms involved the silent information regulator type 1 (SIRT1)-dependent signalling pathway, confirmed by the fact that the SIRT1 inhibitor nicotinamide counteracted the DECM-mediated anti-senescent effect. Collagen type I, rather than fibronectin, partially contributed to the protective effect of decellularized matrix. These findings provide a new strategy of using stem cell-deposited matrix to overcome the challenge of cellular senescence and to facilitate the clinical application of MSCs in regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.