Eradication of cancer stem-like cells (CSLCs) are becoming increasingly an important target for new cancer therapies. The ability to study their behavior in vitro will provide the opportunity for high-throughput testing of more effective treatments. In this study， spheroid-like structures' formation and enrichment of HT29 CSLCs were evaluated on a wool keratin-based substrate as a bio-mimic of natural extracellular matrix (ECM) proteins. The results indicated that culturing on keratin substrate increased spheroid formation ability and radio-/chemoresistance of HT29 cells. Moreover， cell surface expression of CD133 CSLCs' marker and the mRNA level of stemness genes such as Nanog， Oct4， and c-MYC were increased. These data suggest that keratin can potentially be used for spheroid-like structure formation and enrichment of HT29 CSLCs. In addition， it seems that the induction of stemness characteristics on keratin substrate is probably because of the activation of α β integrin signaling pathway. ? 2018 Wiley Periodicals， Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1264-1271， 2019.