Tumor cells utilize inappropriate epithelial-mesenchymal transition (EMT) mechanisms during the invasive process. It is becoming increasingly clear that estradiol (E2) induces breast cancer cell progression and enhances EMT; however, the mechanisms associated with this are unclear. We investigated the role of E2 on the expression and intracellular localization of the tight junction (TJ)-associated proteins, zonula occluden 1 (ZO-1), ZO-1-associated nucleic acid binding (ZONAB), and occludin, on the activation of c-Src and human epidermal growth factor receptor 2 (HER2) expression and cellular migration in the estrogen receptor (ER)-positive breast cancer cell lines, MCF-7 and T47D. We demonstrated that 1 nM E2 elicits c-Src activation after 15 min. The p-Src/ZO-1 complex led to ZO-1 and ZONAB disruption at the TJ and increased expression of HER2 mRNAs. These changes correlate with decreased expression of the epithelial markers occludin and CRB3 and increased synthesis of N-cadherin. This led to increased MCF-7 cell migration induced by E2, even in the presence of a cell proliferation inhibitor. Incubation with ICI 182,780 (Fulvestrant), an ER antagonist, precluded the effects of E2 on c-Src phosphorylation, p-Src/ZO-1 complex formation, ZO-1/ZONAB nuclear translocation, and migration of MCF-7 cells. Our findings suggest that E2 promotes TJ disruption during tumor progression and increases cell motility. We propose a novel pathway where estrogens promote EMT-associated mechanisms that possibly lead to metastasis.