Purpose In our previous studies we showed that upregulating claudin-6 (CLDN6) expression may contribute to preventing breast cancer, and that 17β-estradiol induces a concentration- and time-related effect on CLDN6 mRNA and protein expression in MCF-7 cells. However, the mechanisms of 17β-estradiol regulation of CLDN6 are still unclear. We determined the role of estrogen receptors in the regulation of CLDN6 expression in human breast cancer tissues and a cell line. Methods CLDN6, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) expression in breast cancer tissues were examined using immunohistochemistry. The human breast cancer cell line, MCF-7, which expresses ERα but not ERβ was used. CLDN6 and ERα expression were measured by reverse transcriptase-PCR, Western blotting and immunofluorescent staining. Treatments with propyl pyrazole triol (PPT) and ICI 182, 780 (ICI) were performed. Results The results revealed that CLDN6 expression was related to ERα in breast cancer tissues (p=0.033). PPT, an ERα-selective ligand, upregulated CLDN6 expression at 10-5 mol/L after 24 hours. The effect of PPT on regulating CLDN6 expression in MCF-7 cells was blocked by ICI. Conclusion These findings suggest that Erα reulates CLDN6 expression in breast cancer tissues and that 17β-estradiol induces CLDN6 expression through an ERα pathway in MCF-7 cells.