Background Currently used indicators of iron status have limitations. Hepcidin, a key regulator of iron metabolism, is reduced in iron deficiency. We sought to determine the properties of hepcidin as a diagnostic test of iron deficiency. Design and Methods Sera from female, non-anemic, whole blood donors were analyzed for hepcidin (enzymelinked immunosorbent assay), ferritin, soluble transferrin receptor and C-reactive protein. Iron deficiency was defined as (i) serum ferritin less than 15 ng/mL or (ii) soluble transferrin receptor /log(ferritin) index greater than 3.2 if the C-reactive protein concentration was less than 10 mg/L, or greater than 2.2 if the C-reactive protein concentration was greater than 10 mg/L). Receiver operating characteristic curves were plotted to determine the overall utility and identify optimal cut-points of hepcidin as a test of iron deficiency. Results In 261 blood donors the prevalence of iron deficiency defined by ferritin concentration was 59/261 [22.6 (17.5, 27.7)], whereas defined by soluble transferrin receptor/log(ferritin) index it was 53/261 [20.4 (15.4, 25.2)]. The 95 reference range of hepcidin concentration in the iron-replete population was 8.2-199.7 ng/mL. The area under the receiver operating characteristic curve for hepcidin compared with ferritin concentration less than 15 ng/mL was 0.87 (0.82, 0.92), while that compared with the soluble transferrin receptor /log(ferritin) index was 0.89 (95 CI 0.84, 0.93). For a diagnosis of iron deficiency defined by the soluble transferrin receptor/ log(ferritin) index, hepcidin less than 8 ng/mL had a sensitivity of 41.5 and a specificity of 97.6 , while hepcidin less than 18 ng/mL had a sensitivity of 79.2 and a specificity of 85.6 . Conclusions Serum hepcidin concentration may be a useful indicator of deficient iron stores. Further studies are required to evaluate the role of hepcidin in the diagnosis of iron deficiency in other groups of patients.