Estrogen receptor (ER) plays an important role in several human cancers. Most current ER antagonists bind in the receptor ligand binding pocket and compete for binding with estrogenic ligands. Instead of the traditional approach of targeting estrogen binding to ER, we describe a strategy using a high throughput fluorescence anisotropy microplate assay to identify small molecule inhibitors of ER binding to consensus estrogen response element (cERE) DNA. We identified small molecule inhibitors of ER binding to the fluorescein-labeled (fl)cERE and evaluated their specificity, potency and efficacy. One small molecule, theophylline, 8-[(benzylthio)methyl]- (7CI,8CI) (TPBM), inhibited ER binding to the flcERE (IC₅₀ ~3 M) and inhibited ER-mediated transcription of a stably transfected ERE-containing reporter gene. Inhibition by TPBM was ER specific since progesterone and glucocorticoid receptor transcriptional activity were not significantly inhibited. In tamoxifen-resistant breast cancer cells that overexpress ER, TPBM inhibited 17-estradiol (E₂)-ER (IC₅₀ 9 M) and 4-hydroxytamoxifen-ER-mediated gene expression. Chromatin immunoprecipitation showed TPBM reduced E₂-ER recruitment to an endogenous estrogen-responsive gene. TPBM inhibited E₂-dependent growth of ER positive cancer cells (IC₅₀ 5 M). TPBM is not toxic to cells and doesn’t effect estrogen-independent cell growth. TPBM acts outside of ERs ligand-binding pocket, does not act by chelating the zinc in ERs zinc fingers and differs from known ER inhibitors. Using a simple high throughput screen for inhibitors of ER binding to the cERE, a small molecule inhibitor has been identified that selectively inhibits ER mediated gene expression and estrogen dependent growth of cancer cells.