β-thalassemia major causes ineffective erythropoiesis and chronic anemia and is associated with iron overload due to both transfused iron and increased iron absorption, the latter mediated by suppression of the iron-regulatory hormone hepcidin. We sought to determine whether, in β-thalassemia major, transfusion-mediated inhibition of erythropoiesis dynamically affects hepcidin. We recruited 31 chronically transfused patients with β-thalassemia major and collected samples immediately before and 4 to 8 days after transfusion. Pretransfusion hepcidin was positively correlated with hemoglobin and ferritin and inversely with erythropoiesis. The hepcidin-ferritin ratio indicated hepcidin was relatively suppressed given the degree of iron loading. Posttransfusion, hemoglobin and hepcidin increased, and erythropoietin and growth differentiation factor-15 decreased. By multiple regression, pre- and posttransfusion hepcidin concentrations were both associated positively with hemoglobin, inversely with erythropoiesis, and positively with ferritin. Although men and women had similar pretransfusion hemoglobin, men had significantly increased erythropoiesis and lower hepcidin, received a lower transfusion volume per liter blood volume, and experienced a smaller posttransfusion reduction in erythropoiesis and hepcidin rise. Age of blood was not associated with posttransfusion hemoglobin or ferritin change. Hepcidin levels in patients with β-thalassemia major dynamically reflect competing influences from erythropoiesis, anemia, and iron overload. Measurement of these indices could assist clinical monitoring.