A dose-response experiment was conducted to find the sensitive and consistent biomarker for the estimation of dietary manganese (Mn) requirement and establish the optimal Mn level for broilers fed a practical corn-soybean meal diet from 1 to 21 days of age post-hatching. A total of 480 1-day-old Arbor Acres male chicks were randomly allotted to one of eight treatments with five replicates of 12 birds each and fed diets supplemented with 0, 20, 40, 60, 80, 100, 120, or 140 mg Mn/kg from reagent grade Mn sulfate. Tissue Mn concentrations, manganese-containing superoxide dismutase (MnSOD) activity, and MnSOD mRNA concentration within heart tissue were analyzed at 7, 14, and 21 days of age. Tissue Mn concentrations and heart MnSOD activity showed significant quadratic responses, and heart MnSOD mRNA concentration showed an asymptotic response to dietary supplemental Mn level, respectively. The estimate of dietary Mn for chicks from 1 to 21 days of age was 122-128 for heart Mn concentration, 141-159 for pancreas Mn concentration, 127-138 for liver Mn concentration, and 135-156 mg/kg for heart MnSOD activity, respectively. Heart MnSOD mRNA concentration was a consistent index for the estimation of the Mn requirement of broilers. Based on this index, the estimate of dietary Mn requirement for broilers from 1 to 21 days of age post-hatching was about 130 mg/kg, which was a little more than two times of the current NRC (1994) requirement.