Estrogen (E2) plays a central role in the developmental, metabolic and reproductive functions of both males and females. E2 acts via the estrogen receptor alpha (ERα) to regulate the transcription of genes involved in numerous cellular functions. The E2-dependent engagement of ERα across regulatory regions of the genome, termed “enhancers”, exhibits a high degree of complexity and plasticity. The E2-transcriptional response is defined by pioneer factors, transcription co-factors, posttranslational modifications of ERα, the chromatin environment, and cross talk with other signaling pathways. These inputs collectively define the normal functions of tissues like the mammary gland and the uterus. This mechanism can also provide a selective and aberrant growth advantage in pathological conditions, such as cancer. E2-regulated transcription continues to be an area of great interest in the fields of reproduction and cancer. The goal of these field is to decipher the molecular mechanisms governing ERα transcription to design effective therapeutic strategies for the improvement of clinical care and control of fertility.