Previous global transcriptome and interactome analyses of copper-treated HepG2 cells identified hepatocyte nuclear factor 4α (HNF4α) as a potential master regulator of copper-responsive transcription. Copper exposure caused a decrease in the expression of HNF4α at both mRNA and protein levels, which was accompanied by a decrease in the level of HNF4α binding to its consensus DNA binding sequence. qRT-PCR and RNAi studies demonstrated that changes in HNF4α expression ultimately affected the expressions of its down-stream target genes. Analysis of upstream regulators of HNF4α expression, including p53 and ATF3, showed that copper caused an increase in the steady-state levels of these proteins. These results support a model for copper-responsive transcription in which the metal affects ATF3 expression and stabilizes p53 resulting in the down-regulation of HNF4α expression. In addition, copper may directly affect p53 protein levels. The suppression of HNF4α activity may contribute to the molecular mechanisms underlying the physiological and toxicological consequences of copper toxicity in hepatic-derived cells.