Antiestrogen resistance is a major obstacle to endocrine therapy for breast cancers. Although reduced estrogen receptor-α (ER-α) expression is a known contributing factor to antiestrogen resistance, the mechanisms of ER-α downregulation in antiestrogen resistance are not fully understood. Here, we report that ectopic zinc-finger E-box binding homeobox 1 (ZEB1) is associated with ER-α deficiency in breast cancer cells and thus confers antiestrogen resistance. Mechanistically, ZEB1 represses ER-α transcription by forming a ZEB1/DNA methyltransferase (DNMT)3B/histone deacetylase (HDAC)1 complex on the ER-α promoter, leading to DNA hypermethylation and the silencing of ER-α. Thus, ectopic ZEB1 downregulates ER-α expression and subsequently attenuates cell growth inhibition by antiestrogens, such as tamoxifen and fulvestrant. Notably, the depletion of ZEB1 by RNA interference causes ER-α promoter demethylation, restores ER-α expression, and increases the responsiveness of breast cancer cells to antiestrogen treatment. By studying specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between ZEB1 and ER-α protein expression. Moreover, breast tumors that highly express ZEB1 exhibit ER-α promoter hypermethylation. Using a nude mouse xenograft model, we further confirmed that the downregulation of ZEB1 expression restores the responsiveness of breast cancer cells to antiestrogen therapy in vivo. Therefore, our findings suggest that ZEB1 is a crucial determinant of resistance to antiestrogen therapies in breast cancer.