Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrPC), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrPC in PrP-knock-out (PrP-/-) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with 59Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP-/- mice relative to matched PrP+/+ controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP-/- mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrPC in HepG2 cells increased uptake and storage of ferric iron (Fe3+), not ferrous iron (Fe2+), from the medium, supporting the function of PrPC as a plasma membrane FR. Coexpression of PrPC with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe3+ significantly, and surprisingly, increased the ratio of N-terminally truncated PrPC forms lacking the FR domain relative to full-length PrPC. Together, these observations indicate that PrPC promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed.