Abstract Bromodomain and extra-terminal (BET) family proteins are epigenetic "readers" that are frequently activated in cancer and play an important role in regulating gene expression and oncogenic signaling. Targeting BET proteins using small-molecule inhibitors represents an attractive therapeutic strategy. Several small-molecule BET inhibitors have shown promises in recent preclinical and clinical studies. However, the mechanisms by which BET inhibitors selectively kill cancer cells are not well understood. In this study, we found that the BET inhibitors JQ1 and I-BET151 induce mRNA and protein expression of Death Receptor 5 (DR5) in a variety of colorectal cancer (CRC) cells with different genetic backgrounds. This is an on-target effect as knockdown of the BET family member BRD4 also induces DR5 expression. The induction of DR5 by the BET inhibitors is mediated by C/EBP homologous protein (CHOP) through the endoplasmic reticulum (ER) stress response pathway. Furthermore, knockout of DR5 in colon cancer cells suppresses apoptosis and caspase activation induced by the BET inhibitors, and abrogates their chemosensitization effects in CRC cells. Collectively, our results uncover a key role of DR5 in apoptosis induced by the BET inhibitors in CRC cells, and suggest activation of the death receptor pathway as a novel anticancer mechanism of BET-targeting agents. Citation Format: Xiao Tan, Jingshan Tong, Jian Yu, Liangfang Shen, Lin Zhang. BET inhibitors kill colorectal cancer cells by activating death receptor 5 through the ER stress response pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-191.