Hypoxia occurs during a number of conditions in which altered epithelial proliferation is critical, including tumor development. Microarray analysis of colon-derived epithelial cells revealed a hypoxia-dependent increase in the expression of amphiregulin, an EGF receptor (EGFR) ligand that activates epithelial proliferation and has been associated with the development of colonic tumors. Amphiregulin expression was also induced in tissues from mice exposed to whole animal hypoxia. The hypoxic upregulation of amphiregulin was independent of the classic transcriptional response mediated via hypoxia-inducible factor (HIF)-1alpha. Transfection of HeLa cells with truncated amphiregulin promoter reporter constructs revealed that a 37-bp segment upstream from the TATA box retained hypoxic sensitivity. This sequence contains an evolutionarily conserved cAMP response element (CRE) that constitutively binds the CRE binding protein (CREB). Deletion of the CRE abolished sensitivity to hypoxia. Thus hypoxia promotes intestinal epithelial amphiregulin expression in a CRE-dependent manner, an event that may contribute to increased proliferation. These data also further support a role for CREB as an HIF-independent hypoxia-responsive transcription factor in the regulation of intestinal epithelial gene expression.