Human urine-derived stem cells (hUSCs) are a newly found type of stem cell with a potential for therapeutic application in urology. The aim of this study is to investigate whether hUSCs contribute to cartilage regeneration. Despite their characterization with multi-lineage differentiation capacities, in terms of osteogenesis, adipogenesis and myogenesis, hUSCs do not show the ability to differentiate into chondrocytes. Human bone marrow stromal cells (hBMSCs) are a tissue-specific stem cell for endochondral bone formation; however, repeated-passage hBMSCs have a lower capacity for chondrogenic differentiation. We found that the extracellular matrix (ECM) deposited by hUSCs (UECM) can greatly recharge repeated-passage hBMSCs toward chondrogenic differentiation, a result that might be explained by trophic factors released from hUSCs being immobilized in UECM. We also found that ECM from repeated-passage hBMSCs (BECM) have a limited rejuvenation effect. The Wnt11-mediated noncanonical signaling pathway might be responsible for UECM-mediated hBMSC rejuvenation and subsequent chondrogenic differentiation. Our data indicate that commercially available UECM from young healthy donors might represent a simple and promising approach for autologous hBMSC rejuvenation. This study also provides an excellent model for investigating the effect of trophic factors released by stem cells on tissue regeneration without interference by stem cell differentiation.