Background: The epithelial-mesenchymal transition (EMT) generates cells with properties of stem cells, if that happened, the stem cell should be with mesenchymal property. This study aimed to identify a group of cells with mesenchymal stem cell (MSC)-like characteristics in breast cancer bone metastatic cell line MDA-MB-231, moreover, the relevance between breast cancer stem cells and the EMT was observed. CD105 and CD90, identified as the standards of MSCs, were used for the identification. Methods: The CD105(+)/CD90(+) and CD105(-)/CD90(-) subpopulation of MDA-MB-231 cells were detected and sorted by flow cytometry. MSC-like characteristics in cell proliferation, migration and cell cycle were investigated here by MTT asaay, transwell migration assay, and PI staining respectively. The expression profiles of some stem cell-associated genes were also observed by quantitative real time PCR. Results: Around 0.99% and 90.77% of parental cells were identified as CD105(+)/CD90(+) and CD105(-)/CD90(-) cell subpopulations respectively. The CD105(+)/CD90(+) cells exhibited stronger migratory capacity as compared to parental and CD105(-)/CD90(-) cells, while less CD105(+)/CD90(+) cells were arrested in the S phase. Besides, pluripotent stem cell factors, like Oct-4, Nanog, Klf4 and Sox-2, were all upregulated in CD105(+)/CD90(+) cells, with also proliferation increase, as compared with other two populations. Conclusion: The CD105(+)/CD90(+) subpopulation from breast cancer MDA-MB-231 cells was proven to possess "mesenchymal stem cell-like" characteristics, and its high migratory ability might be associated with EMT. Moreover, using the surface markers of CD105 and CD90 for the identification of MSCs might provide new theoretical basis for the recurrence and metastasis of breast cancer.