HNF4α, a member of the nuclear receptor superfamily, regulates the genes involved in lipid and glucose metabolism. The expression of theRARβgene in the liver of HNF4α knock-out mice was higher versus wildtype controls, whereas oppositely,RARβpromoter activity was 50% reduced by the overexpression of HNF4α in HepG2 cells, and treatment with retinoic acid (RA), a major metabolite of vitamin A, increasedRARβpromoter activity 15-fold. The humanRARβ2promoter contains two DR5 and one DR8 binding motifs, as RA response elements (RARE) proximal to the transcription start site. While DR5 RARE1 was previously reported to be responsive to RARs but not to other nuclear receptors, we show here that mutation in DR5 RARE2 suppresses the promoter response to HNF4α and RARα/RXRα. Mutational analysis of ligand-binding pocket amino acids shown to be critical for fatty acid (FA) binding indicated that RA may interfere with interactions of FA carboxylic acid headgroups with side chains of S190 and R235, and the aliphatic group with I355. These results could explain the partial suppression of HNF4α transcriptional activation toward gene promoters that lack RARE, includingAPOC3andCYP2C9,while conversely, HNF4α may bind to RARE sequences in the promoter of the genes such asCYP26A1andRARβ, activating these genes in the presence of RA. Thus, RA could act as either an antagonist towards HNF4α in genes lacking RAREs, or as an agonist for RARE-containing genes. Overall, RA may interfere with the function of HNF4α and deregulate HNF4α targets genes, including the genes important for lipid and glucose metabolism.