Abstract The Id family of helix-loop-helix (HLH) proteins, Id1, Id2, Id3 and Id4, play a critical role in inhibiting differentiation during mammalian embryogenesis. They function in part by sequestering ubiquitously expressed E protein bHLH transcription factors via direct protein-protein interactions. Various Id proteins are re-expressed in adults in a number of pathologic states including cancer and diseases of the vasculature, where their activity has been shown to be essential for disease progression. The present study describes the solving of the Id1-E47 dimer crystal structure and subsequent development and characterization of a small molecule antagonist of the Id protein family, AGX51. AGX51 was identified in an in silico screen for compounds that could bind a hydrophobic crevice adjacent to the loop region of Id1, highly conserved in the Id family. AGX51 inhibits the endogenous Id1-E protein interaction leading to the degradation of Id1 via ubiquitin-mediated proteolysis. The stability of all four members of the Id family are antagonized by AGX51 leading to a G0-G1 arrest and profound inhibition of viability with no acquired resistance observed in multiple cell lines after continuous exposure to the compound. Administration of AGX51 is well tolerated in mice and phenocopies genetic loss of Id expression analyses: suppression of breast cancer metastases to the lung associated with a reduced mesenchymal-to-epithelial transition, perturbation of the vasculature within the primary tumor, and growth regression of paclitaxel resistant breast tumors in combination with paclitaxel therapy. These studies identify a novel, first-in-class compound capable of antagonizing the activity of a protein family formerly considered undruggable and point to the possible utility of AGX51 in the management of multiple disease processes in patients. Citation Format: Paulina M. Wojnarowicz, Bina Desai, Yvette Chin, Sang Bae Lee, Marta Garcia-Cao, Ouathek Ouerfelli, Guangli Yang, Sijia Xu, Yehuda Goldgur, Meredith A. Miller, Jaideep Chaudhary, William A. Garland, Steven K. Albanese, Rajesh Soni, John Philip, Larry Norton, Neal Rosen, Ronald C. Hendrickson, Xi Kathy Zhou, Antonio Iavarone, Andrew J. Dannenberg, John D. Chodera, Nikola Pavletich, Anna Lasorella, Robert Benezra. A small molecule pan Id protein antagonist shows strong antitumor activity [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 4975. doi:10.1158/1538-7445.AM2017-4975