The duodenal mucosa, cyclically exposed to gastric acid, absorbs dietary iron via duodenal cytochrome b reductase (Dcytb) and the divalent metal ion transporter‐1 (DMT1), upregulated by hypoxia inducible factor‐2α. Since acid increases luminal H2O2 production via the ATP‐P2Y‐Duox2 pathway, we hypothesized that luminal acid induces epithelial hypoxia, affecting the expression of Dcytb and DMT1. We measured epithelial hypoxia by Hypoxyprobe staining on cryostat sections. Pimonidazole was given ip to overnight fasted, freely fed, or omeprazole (OPZ)‐ or catalase (Cat)‐treated rats. The duodenal loop of anesthetized rats was perfused with pH 7 or pH 2.2 saline for 10 min, following pimonidazole iv. Hypoxyprobe staining was heterogeneous in duodenal villous cells: fluorescence intensity was higher in the duodenal villi of fed rats compared to fasting, luminal acid exposure increased Hypoxyprobe staining, compared to pH 7, and Hypoxyprobe staining was decreased in the duodenal villi of OPZ‐and Cat‐treated fed rats. Expression of DMT1 and Dcytb was doubled in the fed group, compared to the fasted group, and inhibited by OPZ treatment. Acid exposure with H2O2 production may induce post‐prandial pO2 fluctuation and maintain the expression level of DMT1 and Dcytb, implicated in OPZ‐induced iron deficiency. VA Merit Review, NIH R01 DK54221