Extracellular matrix (ECM) derived from mesenchymal stem cells regulates antioxidant properties and bone metabolism by providing a favorable extracellular microenvironment. However, its functional role and molecular mechanism in mitochondrial function regulation and aged bone regeneration remain insufficiently elucidated. This proteomic analysis has revealed a greater abundance of proteins supporting mitochondrial function in the young ECM (Y-ECM) secreted by young bone marrow-derived mesenchymal stem cells (BMMSCs) compared to the aged ECM (A-ECM). Further studies demonstrate that Y-ECM significantly rejuvenates mitochondrial energy metabolism in adult BMMSCs (A-BMMSCs) through the promotion of mitochondrial respiratory functions and amelioration of oxidative stress. A-BMMSCs cultured on Y-ECM exhibited enhanced multi-lineage differentiation potentials in vitro and ectopic bone formation in vivo. Mechanistically, silencing of silent information regulator type 3 (SIRT3) gene abolished the protective impact of Y-ECM on A-BMMSCs. Notably, a novel composite biomaterial combining hyaluronic acid methacrylate hydrogel microspheres with Y-ECM is developed, which yielded substantial improvements in the healing of bone defects in an aged rat model. Collectively, these findings underscore the pivotal role of Y-ECM in maintaining mitochondrial redox homeostasis and present a promising therapeutic strategy for the repair of aged bone defects.