Zip14 is a member of the SLC39A zinc transporter family, which is involved in zinc uptake by cells. Up-regulation of Zip14 by IL-6 appears to contribute to the hepatic zinc accumulation and hypozincemia of inflammation. At least three members of the SLC39A family transport other trace elements, such as iron and manganese, in addition to zinc. We analyzed the capability of Zip14 to mediate non-transferrin-bound iron (NTBI) uptake by overexpressing mouse Zip14 in HEK 293H cells and Sf9 insect cells. Zip14 was found to localize to the plasma membrane, and its overexpression increased the uptake of both ⁶⁵Zn and ⁵⁹Fe. Addition of bathophenanthroline sulfonate, a cell-impermeant ferrous iron chelator, inhibited Zip14-mediated iron uptake from ferric citrate, suggesting that iron is taken up by HEK cells as Fe²⁺. Iron uptake by HEK and Sf9 cells expressing Zip14 was inhibited by zinc. Suppression of endogenous Zip14 expression by using Zip14 siRNA reduced the uptake of both iron and zinc by AML12 mouse hepatocytes. Zip14 siRNA treatment also decreased metallothionein mRNA levels, suggesting that compensatory mechanisms were not sufficient to restore intracellular zinc. Collectively, these results indicate that Zip14 can mediate the uptake of zinc and NTBI into cells and that it may play a role in zinc and iron metabolism in hepatocytes, where this transporter is abundantly expressed. Because NTBI is commonly found in plasma of patients with hemochromatosis and transfusional iron overload, Zip14-mediated NTBI uptake may contribute to the hepatic iron loading that characterizes these diseases.