Two experiments were conducted with 28-d-old commercial male broilers to study the kinetics of iron (Fe) absorption and the effect of Fe treatment on divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) mRNA levels in in situ ligated segments from different small intestinal regions of broilers. In Exp. 1, we compared Fe absorption in 3 small intestinal segments at different post-perfusion time points after perfusion with 0.45 m of Fe as Fe sulfate (FeSO ∙ 7HO), and found that the Fe absorption in the duodenum at 30, 45, and 60 min was greater ( < 0.006) than that in the jejunum, and at 60 min, the Fe absorption in the duodenum was greater ( = 0.034) than that in the ileum. In addition, the Fe absorption at 30 min was more than 85.0% of the maximum absorption in each segment. In Exp. 2, a kinetic study of Fe absorption was performed with the duodenal, jejunal, and ileal loops perfused with solutions containing 0 (control), 0.11, 0.22, 0.45, 0.80, 1.79, or 3.58 m of Fe as FeSO 7HO. The Fe concentrations in perfusates were measured at 30 min after perfusion, and in the control group and the group treated with 0.45 m Fe as FeSO 7HO, the DMT1 and FPN1 mRNA levels in the ligated duodenum, jejunum, and ileum were analyzed. The kinetic curves of Fe absorption showed that Fe absorption in the duodenum and jejunum depended on a saturated carrier-mediated process. The maximum absorption rate in the duodenal segment was greater ( < 0.0001) than that in the jejunum (42.75 vs. 8.16 nmol × cm × min), and the Michaelis-Menten constant value was higher ( < 0.0001) in the duodenum than in the jejunum (6.16 vs. 1.31 m). In the ileum, however, the Fe absorption was a non-saturated diffusion process, and the diffusive constant was 3.54 × 10 cm × min. The DMT1 and FPN1 mRNA levels in the duodenum were greater ( < 0.0001) than those in the jejunum and ileum, and greater ( < 0.009) in the jejunum than in the ileum. No differences ( > 0.25) were detected in the DMT1 and FPN1 mRNA levels of the duodenum or jejunum and the DMT1 mRNA level of the ileum between the control and the 0.45 m Fe group, but Fe perfusion increased ( < 0.03) FPN1 mRNA level in the ileum. The above results indicate that the duodenum is the main site of Fe absorption in the small intestine of broilers, and Fe absorption in the duodenum and jejunum is a saturated carrier-mediated process, but a non-saturated diffusion process in the ileum.