Breast cancer is a highly frequent and lethal malignancy which metastasis and relapse frequently associates with the existence of breast cancer stem cells (CSCs). CSCs are undifferentiated, aggressive and highly resistant to therapy, with traits modulated by microenvironmental cells and the extracellular matrix (ECM), a biologically complex and dynamic structure composed mainly by type I collagen (Col-I). Col-I enrichment in the tumor-associated ECM leads to microenvironment stiffness and higher tumor aggressiveness and metastatic potential. While Col-I is also known to induce tumor stemness, it is unknown if such effect is dependent of Col-I density. To answer this question, we evaluated the stemness phenotype of MDA-MB-231 and MCF-7 human breast cancer cells cultured within gels of varying Col-I densities. High Col-I density increased CD44+CD24- breast cancer stem cell (BCSC) immunophenotype but failed to potentiate Col-I fiber alignment, cell self-renewal and clonogenicity in MDA-MB-231 cells. In MCF-7 cells, high Col-I density decreased total levels of variant CD44 (CD44v). Common to both cell types, high Col-I density induced neither markers related to CSC nor those related with mechanically-induced cell response. We conclude that high Col-I density per se is not sufficient to fully develop the BCSC phenotype. © 2020 Valadão et al.