ERα is a central transcription factor, activated by ligand-dependent (estradiol [E2]) or –independent pathways (e.g. cAMP). ERα is expressed in two thirds of all breast tumors (ERα-positive). The final stage of breast cancer is metastasis. In order to better study breast cancer and its progression, the use of three-dimensional (3D) cultures combines both the utility of in vitro models with the advantages of systems closer to physiological conditions and the tumor microenvironment. During this study, ERα responses in different cellular states were evaluated in the ERα+ breast cancer cell lines MDA-MB-134 and MCF-7. The relationship between ERα and an EMT-inducing transcription factor (EMT-TF), ZEB1, was studied when ERα is activated with both ways and between classical 2D and 3D cell cultures. Exogenous ZEB1 expression in ERα+ breast cancer cells increases cell migration and invasion. 2D and 3D cell culture methods delivered significantly different results in comparison.