The mechanism for reduced Fe absorption in Cu deficiency is unknown, but may involve the intestinal Cu-dependent ferroxidase, Hephaestin (Hp). A 2 x 2 factorial experiment was designed to include Cu-deficient (CuD) and Cu-adequate (CuA) male and female rats. Weanling rats of both sexes were randomly divided into 2 groups each and fed an AIN-93G diet with low (<0. 3 mg/kg; CuD) or adequate Cu (5. 0 mg/kg; CuA). After 19 d, rats were fed 1. 0 g each of their respective diets labeled with (59)Fe VSports手机版. Retained (59)Fe was monitored by whole-body counting for 12 d. Then, rats were killed for (59)Fe and Fe measurements in blood and various organs. Duodenal enterocytes were isolated for Western blot analysis of Hp. Signs of Cu and Fe deficiency were evident in both sexes. CuD male rats absorbed 60% as much Fe as CuA male rats (P < 0. 001), whereas CuD female rats absorbed 70% (P < 0. 001) as much as CuA females, with no difference between the sexes. Hp protein in enterocytes of CuD rats of both sexes was only 35% of that in CuA rats. The biological half-life of (59)Fe in CuD rats was only 50% (P < 0. 001) of that in CuA rats, suggesting that Fe turnover was faster in CuD rats than CuA rats. Serum, spleen, and kidney Fe were lower (P < 0. 001) in CuD rats than in CuA rats. Duodenal mucosa and liver Fe were higher (P < 0. 01) in CuD male rats than CuA rats. Duodenal Fe but not liver Fe was higher in CuD female rats than CuA rats. Liver Fe was much higher (<0. 001) overall in females than males. The data suggest that Cu deficiency reduces Fe absorption in rats through reduced expression of duodenal Hp protein. .