Recent studies have shown that overexpression of the transmembrane protein Zrt- and Irt-like protein 14 (Zip14) stimulates the cellular uptake of zinc and nontransferrin-bound iron (NTBI). Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of 55Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe2+) over ferric ion (Fe3+). Zip14-mediated 55Fe2+ uptake was saturable ( K0.5 ≈ 2 μM), temperature-dependent (apparent activation energy, Ea = 15 kcal/mol), pH-sensitive, Ca2+-dependent, and inhibited by Co2+, Mn2+, and Zn2+. HCO3− stimulated 55Fe2+ transport. These properties are in close agreement with those of NTBI uptake in the perfused rat liver and in isolated hepatocytes reported in the literature. Zip14 also mediated the uptake of 109Cd2+, 54Mn2+, and 65Zn2+ but not 64Cu (I or II). 65Zn2+ uptake also was saturable ( K0.5 ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca2+ dependence of Zn2+ transport differed from those of Fe2+ transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.