The transcription factor NRF2 (NFE2L2) is a pivotal activator of genes encoding cytoprotective and detoxifying enzymes that limit the action of cytotoxic therapies in cancer. NRF2 acts by binding antioxidant response elements (ARE) in its target genes, but there is relatively limited knowledge about how it is negatively controlled. Here, we report that retinoic X receptor alpha (RXRa) is a hitherto unrecognized repressor of NRF2. RNAi-mediated knockdown of RXRa increased basal ARE-driven gene expression and induction of ARE-driven genes by the NRF2 activator tert-butylhydroquinone (tBHQ). Conversely, overexpression of RXRa decreased ARE-driven gene expression. Biochemical investigations showed that RXRa interacts physically with NRF2 in cancer cells and in murine small intestine and liver tissues. Furthermore, RXRa bound to ARE sequences in the promoters of NRF2-regulated genes. RXRa loading onto AREs was concomitant with the presence of NRF2, supporting the hypothesis that a direct interaction between the two proteins on gene promoters accounts for the antagonism of ARE-driven gene expression. Mutation analyses revealed that interaction between the two transcription factors involves the DNA-binding domain of RXRa and a region comprising amino acids 209-316 in human NRF2 that had not been defined functionally, but that we now designate as the NRF2-ECH homology (Neh) 7 domain. In non-small cell lung cancer cells where NRF2 levels are elevated, RXRa expression downregulated NRF2 and sensitized cells to the cytotoxic effects of therapeutic drugs. In summary, our findings show that RXRa diminishes cytoprotection by NRF2 by binding directly to the newly defined Neh7 domain in NRF2. Cancer Res; 73(10); 3097-108. ©2013 AACR.