Background: Self-assembling peptide nanofiber scaffolds have been shown to be a permissive biological material for tissue repair, cell proliferation, differentiation, etc. Recently, a subpopulation (CD44(+)/CD24(-)) of breast cancer cells has been reported to have stem/progenitor cell properties. The aim of this study was to investigate whether this subpopulation of cancer cells have different phenotypes in self-assembling COCH3-RADARADARADARADA-CONH2 (RADA16) peptide nanofiber scaffold compared with Matrigel (R) (BD Biosciences, Two Oak Park, Bedford, MA, USA) and collagen I. Methods: CD44 and CD24 expression was determined by flow cytometry. Cell proliferation was measured by 5-bromo-2'-deoxyuridine assay and DNA content measurement. Immunostaining was used to indicate the morphologies of cells in three-dimensional (3D) cultures of different scaffolds and the localization of beta-catenin in the colonies. Western blot was used to determine the expression of signaling proteins. In vitro migration assay and inoculation into nude mice were used to evaluate invasion and tumorigenesis in vivo. Results: The breast cancer cell line MDA-MB-435S contained a high percentage (>99%) of CD44(+)/CD24(-) cells, which exhibited phenotypic reversion in 3D RADA16 nanofiber scaffold compared with collagen I and Matrigel. The newly formed reverted acini-like colonies reassembled a basement membrane and reorganized their cytoskeletons. At the same time, cells cultured and embedded in RADA16 peptide scaffold exhibited growth arrest. Also, they exhibited different migration potential, which links their migration ability with their cellular morphology. Consistent with studies in vitro, the in vivo tumor formation assay further supported of the functional changes caused by the reversion in 3D RADA16 culture. Expression levels of intercellular surface adhesion molecule-1 were upregulated in cells cultured in RADA16 scaffolds, and the NF-kappa B inhibitor pyrrolidine dithiocarbamate could inhibit RADA16-induced upregulation of intercellular surface adhesion molecule-1 and the phenotype reversion of MDA-MB-453S cells. Conclusion: Culturing a CD44(+)/CD24(-)-enriched breast cancer cell population in 3D RADA16 peptide nanofiber scaffold led to a significant phenotypic reversion compared with Matrigel and collagen I.