ABSTRACT: The objective of this study was to determine the effect of organic and inorganic Fe sources on the Fe absorption and gene expression of Fe and amino acid transporters in the ligated jejunal and ileal segments of broilers. The in situ ligated jejunal and ileal loops from Fe-deficient broiler chicks (28-d-old) were perfused with Fe solutions containing 0, 3.58, or 7.16 mM Fe from one of the following Fe sources: Fe sulfate (FeSO4?7H2O), the mixtures of FeSO4?7H2O with either Met or Gly, Fe-Gly chelate, or three Fe-amino acid or protein chelates with weak, moderate or extremely strong chelation strengths (Fe-Met W, Fe-Pro M, or Fe-Pro ES), respectively, for up to 30 min. Iron absorption was increased (P < 0.0001) as the perfused Fe concentrations increased, and no differences (P > 0.07) were detected in the Fe absorption between the jejunum and ileum. Regardless of intestinal segments, Fe absorption was higher (P < 0.006) for Fe-Pro ES and Fe-Pro M than for FeSO4·7H2O, and for Fe-Pro ES than for Fe-Met W. Glycine but not Met supplementation increased (P < 0.03) the absorption of Fe as FeSO4. Regardless of Fe source, Fe addition inhibited (P < 0.05) the mRNA expressions of divalent metal transporter 1 (DMT1) in the jejunum and ileum, but enhanced (P < 0.05) the mRNA expressions of l-type amino transporter 1 (LAT1) and B0-type amino acid transporter 1 (B0AT1) in the jejunum and ileum. No differences (P > 0.05) among different Fe sources were observed in the mRNA expression levels of Fe and amino acid transporters in both the jejunum and the ileum. The mRNA expression levels of DMT1, ferroportin 1, B0AT1, or y+LAT1 were higher (P < 0.0001), but those of excitatory amino acid transporter 3, LAT1, or y+l-type amino transporter 2 were lower (P < 0.04) in the jejunum than in the ileum. The supplementation of inorganic or organic Fe had no effect (P > 0.14) on the protein expression levels of DMT1 and FPN1 in the jejunum and ileum. The above results indicate that organic Fe sources with stronger chelation strengths showed higher Fe absorption in the jejunum and ileum of broiler chicks. Glycine was more effective in facilitating Fe absorption than Met as a ligand. The mRNA expressions of Fe and amino acid transporters in the jejunum were different from those in the ileum. The DMT1, LAT1, and B0AT1 might be involved in the Fe absorption in the jejunum or ileum of broilers.