Iron homeostasis-related genes (e.g., Dmt1 and Dcytb) are upregulated by hypoxia-inducible factor 2α (HIF2α) during iron deficiency in the mammalian intestine. Menkes copper ATPase (Atp7a) gene expression is also strongly induced in the duodenum of iron-deficient rats. The current study was thus designed to test the hypothesis that Atp7a is regulated by HIF2α. Rat intestinal epithelial (IEC-6) cells were utilized to model the intestinal epithelium, and CoCl(2) and 1% O(2) were applied to mimic hypoxia in vitro. Both treatments significantly increased endogenous Atp7a mRNA levels; mRNA induction with CoCl(2) treatment was blunted by a transcriptional inhibitor. The rat Atp7a promoter was thus cloned and studied. Various sized promoter constructs were inserted into a luciferase reporter vector and transfected into cells. A -224/+88 bp construct had full activity and was induced by CoCl(2); this promoter fragment was thus utilized for subsequent analyses. Interestingly, this region contains three phylogenetically conserved, putative hypoxia response elements (HRE; 5'-NCGTGN-3'). It was further noted that HIF2α overexpression caused a significant upregulation of promoter activity while HIF1α overexpression had little effect. To determine whether Atp7a is a direct HIF target, three putative HREs were deleted individually or in combination; all were shown to be essential for transcriptional induction. Chromatin immunoprecipitation studies also demonstrated that HIF2α binds to the Atp7a promoter region. Lastly, Atp7a and HIF2α protein levels were shown to be increased by both treatments. In conclusion, the Atp7a gene is upregulated by direct interaction with HIF2α, demonstrating coordinate regulation with genes related to intestinal iron homeostasis.