Abstract Background Use of serum hepcidin measurements in pediatrics would benefit from standardized age‐ and sex‐specific reference ranges in children, in order to enable the establishment of clinical decision limits that are universally applicable. Procedure We measured serum hepcidin‐25 levels in 266 healthy Dutch children aged 0.3‐17 years, using an isotope dilution mass spectrometry assay, standardized with our commutable secondary reference material (RM), assigned by a candidate primary RM. Results We constructed age‐ and sex‐specific values for serum hepcidin and its ratio with ferritin and transferrin saturation (TSAT). Serum hepcidin levels and hepcidin/ferritin and TSAT/hepcidin ratios were similar for both sexes. Serum hepcidin and hepcidin/ferritin ratio substantially declined after the age of 12 years and TSAT/hepcidin ratio gradually increased with increasing age. Serum hepcidin values for Dutch children <12 years (n = 170) and >12 years (n = 96) were 1.9 nmol/L (median); 0.1‐13.1 nmol/L (p2.5‐p97.5) and 0.9 nmol/L; 0.0‐9.1 nmol/L, respectively. Serum ferritin was the most significant correlate of serum hepcidin in our study population, explaining 15.1% and 7.9% of variance in males and females, respectively. Multivariable linear regression analysis including age, blood sampling time, iron parameters, ALT, CRP, and body mass index as independent variables showed a statistically significant negative association between age as a dichotomous variable (≤12 vs >12 years) and log‐transformed serum hepcidin levels in both sexes. Conclusions We demonstrate that serum hepcidin relative to indicators of body iron is age dependent in children, suggesting that the set point of serum hepcidin relative to stored and circulating iron changes during childhood.