Phytoestrogens have been investigated for their potential anti-tumorigenic effects in various cancers including breast cancer. Emodin being a phytoestrogen shows anti-carcinogenic properties especially in estrogen receptor positive (ER+) breast cancers. The aim of this study is to identify the molecular mechanism and related biological pathways in both (ER+) MCF-7 and (ER–) MDA-MB-231 breast cancer cell lines upon Emodin treatment via microarray analysis in order to find out therapeutic biomarkers. In both cell lines, first differentially expressed genes were identified, then gene ontology and functional pathway enrichment analyses were performed. Genes regulated through multiple pathways were studied together with literature and a gene cluster was determined for each cell line. Further GeneMANIA and STRING databases were used to study the interactions within the related gene clusters. The results showed that, the genes which are related to cell cycle were significantly regulated in both cell lines. Also, Forkhead Box O1-related genes were found to be prominent in MCF-7 cells. In MDA-MB-231 cells, spindle attachment checkpoint mechanism-related genes were regulated, remarkably. As a result, novel gene regulations reported in this study in response to Emodin will give more information about its metabolism and antiproliferative effect, especially in ER + cells.