Background: Iron homeostasis proved to be regulated by hepatic peptide hormone "Hepcidin". Iron homeostasis is disturbed in beta-thalassemia and results in iron overload with or without transfusion dependence.Aim: to investigate serum hepcidin level and its potential usefulness in the diagnosis of iron overload in children with beta thalassemia major and intermedia as well as to assess the relationship between hepcidin level and various clinical and laboratory characteristics of recruited patients. Methodology: A prospective study conducted at the Hematology Clinic, El-Monira Children's Hospital, Cairo University. Thirty thalassemia intermedia (TI group), 30 thalassemia major (TM group) and 60 healthy -age and sex- matched subjects were enrolled. All patients were subjected to history taking and complete physical examination. Complete blood count, liver function tests, serum bilirubin (total, direct), serum ferritin and serum hepcidin level was measured by Human Hepcidin, ELISA Kit.Results: Mean serum hepcidin in healthy children was 0.4 ng/ml (0.19 to 0.89 ng/ml) and it was 3.1 ng/ml in TM group and 1.4 ng/ml in TI group (p<0.001). TI group had lower hepcidin compared to TM group (p=0.034). Median hepcidin was significantly higher among those with higher number of total transfusions (p=0.01) and among chelated patients (p=0.002). Among the control group, serum hepcidin didn't correlate with age (r=0.225, p=0.084), but it correlated positively with age, disease duration, transfusion frequency, total number of transfusions, total bilirubin, and serum ferritin in β-thalassemia patients. TM group had higher level of serum ferritin compared to TI group (p<0.001). Hepcidin/ ferritin ratio was <1 in all cases and was comparable between TM and TI patients (p=0.4). Well-chelated patients had lower s hepcidin compared to poor-chelated patients (p<0.05), but Hepcidin/ ferritin ratio was comparable between both groups (p>0.05). Total hemoglobin and serum ferritin were significantly related to serum hepcidin; hepcidin tends to increase by 0.514 ng/ml with each 1g/dl rise in hb (p=0.023) and tends to increase by 0.002 ng/ml with each 1ng/ml rise in serum ferritin (p=0.002), after adjusting for age, sex, all transfusions, chelation and HU therapy. Our data showed that iron overload (SF≥1500 ng/ml) was independantly associated with thalassemia major (p=0.001) and elevated serum hepcidin (p=0.02), however, when significant predictors were considered together, only thalassemia major retained its significance as predictor of iron overload (p=0.022). However, the overall predictability of hepcidin in severe iron overload was statistically significant when compared to hepcidin to ferritin ratio. Conclusion: Serum hepcidin is generally elevated in B-thalassemia patients in the pediatric age group, but this elevation is more evident in TM patients mostly due to the regular transfusion therapy. Serum hepcidin production may be more sensitive to elevations in the total hemoglobin and serum ferritin rather than age, sex or concomitant medications. Serum hepcidin can be adopted as an indicator of severe iron overload.