The relationship between iron and β cell dysfunction has long been recognized as individuals with iron overload display an increased incidence of diabetes. This link is usually attributed to the accumulation of excess iron in β cells leading to cellular damage and impaired function. Yet, the molecular mechanism(s) by which human β cells take up iron has not been determined. In the present study, we assessed the contribution of the metal-ion transporters ZIP14, ZIP8, and DMT1 to iron uptake by human β cells. Iron was provided to the cells as non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload and is a major contributor to tissue iron loading. We found that overexpression of ZIP14 and ZIP8, but not DMT1, resulted in increased NTBI uptake by βlox5 cells, a human β cell line. Conversely, siRNA-mediated knockdown of ZIP14, but not ZIP8, resulted in 50% lower NTBI uptake in βlox5 cells. In primary human islets, knockdown of ZIP14 also reduced NTBI uptake by 50%. Immunofluorescence analysis of islets from human pancreatic sections localized ZIP14 and DMT1 nearly exclusively to β cells. Studies in primary human islets suggest that ZIP14 protein levels do not vary with iron status or treatment with IL-1β. Collectively, these observations identify ZIP14 as a major contributor to NTBI uptake by β cells and suggest differential regulation of ZIP14 in primary human islets compared to other cell types such as hepatocytes.