The expression of estrogen receptor α (ER) in breast cancers may be indicative of a favorable prognosis and most of these cancers respond to anti-estrogens or aromatase inhibitors. However, ER-positive (ER+) breast cancers receiving anti-hormone and/or chemotherapy sometimes lose their ER expression, which leads to the evolution of the disease to higher aggressiveness and drug resistance. In the present study, an ER-modified signature (EMS) was developed from the expression profile of a chemoresistant MCF-7 breast cancer cell line that lost ER expression during long-term treatment with a chemotherapeutic agent. The EMS could discriminate the ER-negative (ER-) breast cancer cells from the ER+ ones, which included seven pathways essential for the ER- cell development. Furthermore, the EMS indicated a more malignant subgroup of the ER- cells by discriminating the chemoresistant ER- cells from the chemosensitive ones. In addition, the classified chemoresistant ER- patients demonstrated worse prognosis. In conclusion, we developed a new method to discriminate subgroups of ER- breast cancer cells.