Beta-thalassemia major (BTM) is associated with significant morbidity and mortality due to iron overload resulting from ineffective erythropoiesis and repeated blood transfusions. The level of hepcidin, a regulator of iron homeostasis, is influenced by anemia and iron overload, both of which are present in children with BTM and have an opposing effect on hepcidin expression. This study aimed to assess the influence of iron overload and enhanced erythropoiesis on the levels of serum hepcidin in multitransfused patients with BTM. Complete blood counts, serum iron (SI), total iron binding capacity (TIBC), percent transferrin saturation (%TS), serum ferritin (SF), serum transferrin receptors (sTfR) and serum hepcidin were measured in 52 patients with BTM and 35 controls. SF and sTfR were significantly (p<0.001) elevated in patients with BTM as compared to controls. Serum hepcidin was significantly (p<0.001) higher in patients (28.3±3.2 ng/ml) as compared to controls (8.3±4.6 ng/ml). A negative correlation was seen between serum hepcidin and SI, TIBC and %TS. No correlation was observed between serum hepcidin and SF as also sTfR. The mean serum hepcidin/ferritin ratio was significantly (p<0.001) lower in patients as compared to controls. The lack of correlation between hepcidin and SF as also sTfR, suggests that in BTM iron stores and erythropoietic activity do not play a role in hepcidin expression. The hepcidin/ferritin ratio was <1 in patients indicating a suppression of hepcidin relative to the degree of iron overload.