Hexavalent chromium [Cr(VI)] induces hematological signs of microcytic anemia in rodents. Considering that Cr(VI) can oxidize ferrous (Fe²⁺) to ferric (Fe³⁺) iron, and that only the former is transported across the duodenum, we hypothesize that, at high concentrations, Cr(VI) oxidizes Fe²⁺ in the lumen of the small intestine and perturbs iron absorption. Herein we report that 90-day exposure to Cr(VI) in drinking water resulted in dose-dependent decreases in Fe levels in the duodenum, liver, serum, and bone marrow. Toxicogenomic analyses from the duodenum indicate responses consistent with Fe deficiency, including significant induction of divalent metal transporter 1 (DMT1, Slc11a2) and transferrin receptor 1 (TFR1, Tfr1). In addition, at ⩾20 mg Cr(VI)/L in drinking water, Cr RBC:plasma ratios in rats were increased and exceeded unity, indicating saturation of reductive capacity and intracellular absorption of Cr(VI) into red blood cells (RBCs). These effects occurred in both species but were generally more severe in rats. These data suggest that high concentrations of Cr(VI) in drinking limit Fe absorption and alter iron homeostasis. Furthermore, some effects observed at high doses in recent Cr(VI) chronic and subchronic bioassays may be explained, at least in part, by iron deficiency and disruption of homeostasis.