Abstract Estrogen receptor alpha (ERα) is a ligand-activated nuclear receptor that regulates proliferation and differentiation in mammary epithelial cells. ERα activity is likely dependent on the actions of pioneer factors and H3K4 methyltransferases which can establish a genomic landscape permissive for ERα binding. Here, we identify the H3K4 methyltransferase KMT2C as essential for ERα activity in mammary gland development and ER+ breast cancer growth. KMT2C suppression decreases estrogen-dependent gene expression and causes H3K4me1 loss at ERα target gene enhancers. Consequently, KMT2C loss selectively suppresses estrogen-driven breast cancer proliferation. Moreover, mammary-specific Kmt2c knockout mice have defects in pubertal ductal formation similar to Esr1 deficient mice. Although KMT2C loss disrupts estrogen-driven proliferation, it conversely promotes tumor outgrowth under hormone-depleted conditions. Consistent with this, gene expression signatures of KMT2C loss are associated with poor outcomes. We conclude that KMT2C is a key regulator of ERα activity whose loss uncouples mammary phenotypes from hormone availability. Citation Format: Kinisha Gala, Amit Sinha, Francisco Sanchez-Vega, Young Rock Chung, James Hseih, Michael Berger, Nikolaus Schultz, Alessandro Pastore, Omar Abdel-Wahab, Sarat Chandarlapaty. KMT2C directs estrogen receptor activity in normal and transformed mammary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5500. doi:10.1158/1538-7445.AM2017-5500