BACKGROUND: Human subpatellar fat pad is a kind of waste, which is often removed in total knee arthroplasty. If it is fully utilized, it can be used as a source of mesenchymal stem cells. OBJECTIVE: To explore the methods of isolation, culture and identification of human infrapatellar fat pad derived stem cells in vitro, to induce chondrogenic differentiation under specific conditions, and to explore the feasibility of being used as seed cells for cartilage tissue engineering. METHODS: The discarded infrapatellar fat pad from eight patients undergoing the total knee arthroplasty was obtained and human infrapatellar fat pad derived stem cells were isolated and cultured according to trypsin digestion, type I collagenase digestion and differential adherence methods. After being cultured to the third generation in vitro, cell morphology observation, cell growth curve and cell surface antigen analysis were performed. In addition, chondrogenic differentiation was induced, and human infrapatellar fat pad derived stem cells were identified by immunohistochemistry, toluidine blue staining and RT-qPCR. RESULTS AND CONCLUSION: (1) Isolated, purified and cultured human infrapatellar fat pad derived stem cells showed spindle-shaped adherent growth. The growth curve of the third generation of human infrapatellar fat pad derived stem cells was in the shape of “S”. Cells were positive for CD44 and CD105, while negative for CD45. (2) After 14 days of induction, immunohistochemical staining and toluidine blue staining were positive in the differentiation group, and the relative gene expression levels of ACAN, BMP-2, COL2A1 and SOX-9 were significantly higher than those in the blank control group. (3) This study confirmed that mesenchymal stem cells with differentiation potential could be isolated from infrapatellar fat pad, which had strong proliferation ability and chondrogenic differentiation ability in vitro, and were expected to become ideal seed cells for cartilage tissue engineering. © 2021, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.