Primary cell culture in vitro suffers from cellular senescence. We hypothesized that expansion on decellularized extracellular matrix (dECM) deposited by simian virus 40 large T antigen (SV40 LT) transduced autologous infrapatellar fat pad stem cells (IPFSCs) could rejuvenate high-passage IPFSCs in both proliferation and chondrogenic differentiation. In the study, we found that SV40 LT transduced IPFSCs exhibited increased proliferation and adipogenic potential but decreased chondrogenic potential. Expansion on dECMs deposited by passage 5 IPFSCs yielded IPFSCs with dramatically increased proliferation and chondrogenic differentiation capacity; however, this enhanced capacity diminished if IPFSCs were grown on dECM deposited by passage 15 IPFSCs. Interestingly, expansion on dECM deposited by SV40 LT transduced IPFSCs yielded IPFSCs with enhanced proliferation and chondrogenic capacity but decreased adipogenic potential, particularly for the dECM group derived from SV40 LT transduced passage 15 cells. Our immunofluorescence staining and proteomics data identify matrix components such as basement membrane proteins top candidates for matrix mediated IPFSC rejuvenation. Both cell proliferation and differentiation were endorsed by transcripts measured by RNASeq during the process. This study provides a promising model for in-depth investigation of matrix protein influence on surrounding stem cell differentiation.