The organophosphorus chlorpyrifos (CPF) is currently classified as an Endocrine Disruptor (EDs). EDs have been associated with resistance to endocrine therapy in breast cancer and loss of Estrogen Receptor α (ERα) expression is critical in this process. It is postulated that histone deacetylase 1 (HDAC1) interacts with ERα and suppresses ERα transcriptional activity. Additionally, Cancer Stem Cells (CSC) show a self-renewal capacity and differentiation potential that contribute to tumor progression and therapy resistance. CSC are characterized by the expression of stem cell markers such as OCT4, SOX2, Nanog, ALDH1A1 and CD44+/CD24-. In this study, we investigated whether CPF can induce mechanisms associated with resistance to antiestrogen therapy. Our experiments were performed using MCF-7 cell line. We analyzed if CPF (0.05 and 50 µM) induces CSC proliferation by mammosphere assay and/or CSC markes (CD44, CD24, Oct4 and Nanog) ERα, HDAC1 and the co-repressor SMRT expression in monolayer cells and mammospheres by RT-qPCR. CPF at 0.05 µM increases the subpopulation of CSC derived from MCF-7 cells (p<0.05), decreases ERα and HDAC1 and increases SMRT mRNA expression (p<0.05) in monolayer-grown cells. CPF 50 µM decreases SMRT (p<0.05) and CD24 (p<0.01) mRNA levels in these conditions. In mammospheres, both CPF concentrations induce an decreased ERS1 (p<0.001) and HDAC1 (p<0.001) expression, while only CPF 0.05 µM decreased SMRT (p<0.01) levels. We observed that CPF 0.05 upregulates CD44 (p<0.01) and Oct4 (p<0.001) and downregulates CD24 (p<0.01) expression. CPF 50 µM enhances the expression of Oct4 (p<0.01) and Nanog (p<0.05). Our study demonstrates that CPF is able to increase CSC subpopulation and can modulate the expression of CSC markers and molecular targets involved in antiestrogen therapy resistance. Therefore, CPF exposure could affect the outcome of breast cancer patients treated with endocrine therapy.