Background & Aims The surface of the intestinal mucosa is particularly prone to hypoxia-induced inflammation. Previous studies implicated signaling via extracellular adenosine in endogenous attenuation of intestinal inflammation; we investigated whether epithelial adenosine transport could reduce hypoxia-induced inflammation of the mucosa. Methods We performed in vitro studies of epithelial adenosine uptake and nucleoside transport using cultured epithelial cells. In vivo studies of ambient hypoxia levels were performed using mice with conditional loss of hypoxia-inducible factor (HIF)-α expression in the colon. Results Studies of epithelial adenosine transport under hypoxic conditions showed that extracellular adenosine uptake occurs mainly at the apical surface of epithelial cells and is attenuated by hypoxia. Subsequent transcriptional studies suggested high expression levels of the equilibrative nucleoside transporter-2 (ENT2) in human epithelial cells and revealed ENT2 repression during hypoxia. Studies with promoter constructs, including site-directed mutagenesis, transcription factor binding assays, and HIF loss and gain of function showed a central role of HIF-1α in transcriptional repression of ENT2 during hypoxia. Similarly, transcriptional repression of ENT2 by ambient hypoxia was abolished in conditional HIF-1α mutant mice in vivo. Functional studies using RNA interference showed that loss of epithelial ENT2 was associated with reduced adenosine uptake in vitro, whereas pharmacologic inhibition of ENT2 attenuated hypoxia-induced inflammation of the mucosa in vivo. Conclusions HIF-1α–dependent repression of ENT2 increases mucosal adenosine signaling and attenuates hypoxia-associated inflammation of the intestine.