Background: In vitro 3-dimensional spheroid culture has been widely used as model to enrich CD44+CD24dim/- cancer stem cells (CSC) with high ALDH1 activity. Although CD24+subpopulation was known to be present in 3D spheroids and may influence cancer drug therapies, its characteristics and CSC properties were not well defined. Methods: In this study, CD24+ population from the MCF-7 spheroid was sorted and subjected to spheroid formation test, stem cell markers immunofluorescence, invasion and migration test as well as microRNA expression profiling. Results: Sorted MCF-7 CD24+ cells from primary spheroids were able to reform its 3D spheroid shape after 7 days in non-adherent culture conditions. In contrast to the primary spheroids, the expression of SOX-2, CD44, CD49f and Nanog were dim in MCF-7 CD24+ cells. Remarkably, MCF-7 CD24+ cells were found to show high expression of ALDH1 protein which may have resulted in these cells exhibiting higher resistance against doxorubicin and cisplatin when compared to that of the parental cells. Moreover, microRNA profiling has shown that the absence of cancer stem cell properties were consistent with the downregulation of major cancer stem cells related pathways including Hedgehog, Wnt and MAPK signalling pathways. However, the upregulated pathways such as adherans junctions, focal adhesion and tight junction suggest that CD24+ cells were probably at an epithelial-like state of cell transition. Conclusion: In conclusion, neglected CD24+ cells in MCF-7 spheroid did not exhibit typical breast CSCs properties. The presence of miRNAs and their analysed pathways suggested that these cells could be a distinct intermediate cell state in breast CSCs.