AbstractHypoxia has been intensively investigated over the past several decades based on the observations that hypoxic tumors are more resistant to therapy and have a worse prognosis. Previously, we reported that N-myc downstream-regulated gene 1 (NDRG1) is strongly up-regulated under hypoxia and may play an important role in tumor adaptation to fluctuating oxygen concentrations. However, the regulatory mechanism ofNDRG1under hypoxia remains elusive. Therefore, the purpose of this study was to identify the transcription factors that regulateNDRG1and to investigate the functional roles ofNDRG1in hypoxia. We showed that binding sites of aryl hydrocarbon receptor (AHR) were predicted in theNDRG1promoter. Nuclear AHR was up-regulated in the presence of cobalt and hypoxia. AHR translocated to nuclei and bound between base pairs −412 and −388 of theNDRG1promoter in hypoxia. Moreover, hypoxia-mimetic induction ofNDRG1was attenuated by knockdown of AHR expression. Also, overexpression of AHR facilitated cell proliferation and migration via up-regulation ofNDRG1. These results showed for the first time that AHR positively regulatesNDRG1transcription through an AHR binding site by way of hypoxia-mimetic signaling, which may lead to development of a specific therapeutic regimen to prevent tumor malignancy under hypoxia.