NR4A1 (Nur77, TR3) is an orphan nuclear receptor that is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity. RNAi interference of NR4A1 expression in Panc-1 cells induced apoptosis and subsequent proteomic analysis revealed the induction of several markers of endoplasmic reticulum (ER) stress including glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein-homologous protein (CHOP), and activating transcription factor-4 (ATF-4). Treatment of pancreatic cancer cells with the NR4A1 antagonist 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH), gave similar results. Moreover, both NR4A1 knockdown and DIM-C-pPhOH induced reactive oxygen species (ROS), and induction of ROS and ER stress by these agents was attenuated after co-treatment with antioxidants. Manipulation of NR4A1 expression coupled with gene expression profiling identified a number of ROS metabolism transcripts regulated by NR4A1. Knockdown of one of these transcripts, thioredoxin domain containing 5 (TXNDC5), recapitulated the elevated ROS and ER stress; thus, demonstrating that NR4A1 regulates levels of ER stress and ROS in pancreatic cancer cells to facilitate cell proliferation and survival. Finally, inactivation of NR4A1 by knockdown or DIM-C-pPhOH decreased TXNDC5, resulting in activation of ROS/ER stress and pro-apoptotic pathways.