Hypoxia upregulates the hypoxia-inducible factor (HIF) pathway and the endoplasmic reticulum (ER) stress signal, unfolded protein response (UPR). The cross talk of both signals affects the pathogenic alteration by hypoxia. Here we showed that ER stress induced by tunicamycin or thapsigargin suppressed inducible (CoCl(2) or hypoxia) transcription of erythropoietin (EPO), a representative HIF target gene, in HepG2. This suppression was inversely correlated with UPR activation, as estimated by expression of the UPR regulator glucose-regulated protein 78, and restored by an ER stress inhibitor, salubrinal, in association with normalization of the UPR state. Importantly, the decreased EPO expression was also observed in HepG2 overexpressing UPR activating transcription factor (ATF)4. Overexpression of mutated ATF4 that lacks the transcriptional activity did not alter EPO transcriptional regulation. Transcriptional activity of the EPO 3'-enhancer, which is mainly regulated by HIF, was abolished by both ER stressors and ATF4 overexpression, while nuclear HIF accumulation or expression of other HIF target genes was not suppressed by ER stress. Chromatin immunoprecipitation analysis identified a novel ATF4 binding site (TGACCTCT) within the EPO 3'-enhancer region, suggesting a distinct role for ATF4 in UPR-dependent suppression of the enhancer. Induction of ER stress in rat liver and kidney by tunicamycin decreased the hepatic and renal mRNA and plasma level of EPO. Collectively, ER stress selectively impairs the transcriptional activity of EPO but not of other HIF target genes. This effect is mediated by suppression of EPO 3'-enhancer activity via ATF4 without any direct effect on HIF, indicating that UPR contributes to oxygen-sensing regulation of EPO.