The pathogenesis of diabetes associated with hemochromatosis is not known. We therefore examined glucose homeostasis and β-cell function in mouse models of hemochromatosis. Mice with targeted deletion of the hemochromatosis gene (Hfe^−/−) on the 129/Sv genetic background exhibited a 72% increase in iron content in the islets of Langerhans compared with wild-type controls. Insulin content was decreased in Hfe^−/− mice by 35%/pancreas and 25%/islet. Comparable decreases were seen in the mRNA levels of β-cell-specific markers, ins1, ins2, and glucose transporter 2. By 6–8 months, islets from Hfe^−/− mice were 45% smaller, associated with increased staining for activated caspase 3 and terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling. Islets from Hfe^−/− mice were also desensitized to glucose, with half-maximal stimulation of insulin secretion seen at 16.7 ± 0.9 mm glucose in perifused islets from Hfe^−/− mice compared with 13.1 ± 0.6 mm glucose in wild-type animals. Carbonyl protein modification, a marker for oxidative stress, was increased by 58% in Hfe^−/− islets. Despite decreased islet size, Hfe^−/− mice exhibited enhanced glucose tolerance. Fasting serum insulin levels were comparable between Hfe^−/− and Hfe^+/+ mice, but were 48% lower in the Hfe^−/− mice 30 min after challenge. Similar results were seen in mice carrying an Hfe mutation analogous to the common human mutation (C282Y) and in mice fed excess dietary iron. Hfe^−/−mice on the C57BL6 background exhibited decreased glucose tolerance at 10–12 months due to an inability to increase insulin levels as they aged. We conclude that iron excess results in β-cell oxidant stress and decreased insulin secretory capacity secondary to β-cell apoptosis and desensitization of glucose-induced insulin secretion. This abnormality alone, however, is insufficient to cause diabetes.