Abnormal iron metabolism can cause oxidative stress in broilers, and transport stress (TS) may potentially influence iron metabolism. However, the mechanisms by which TS affects iron metabolism are unclear. This study used quantitative proteome analysis based on tandem mass tag (TMT) to investigate the effects of TS on liver iron metabolism in broilers. Broilers (n = 24) reared under the same conditions were selected randomly into the transported group for 4 h (T2) and non-transported group (T1). Results showed that the serum iron level and total iron-binding capacity of broilers in the T2 were significantly higher than those in the T1 (p < 0.05). The liver iron content of broilers in the T2 (0.498 ± 0.058 mg·gprot⁻¹) was significantly higher than that in the T1 (0.357 ± 0.035 mg·gprot⁻¹), and the iron-stained sections showed that TS caused the enrichment of iron in the liver. We identified 1139 differentially expressed proteins (DEPs). Twelve DEPs associated with iron metabolism were identified, of which eight were up-regulated, and four were down-regulated in T2 compared with T1. Prediction of the protein interaction network for DEPs showed that FTH1, IREB2, and HEPH play vital roles in this network. The results provide new insights into the effects of TS on broilers’ liver iron metabolism.

• Metabolomics and proteomics analysis of exudate were used to characterize the effect of iron metabolism on beef oxidation. • Ferroptosis was the major iron metabolic pathway during beef ageing. • Disturbed iron metabolism was dependent on free iron accumulation. • Free iron induced oxidative stress in muscle tissue through the Fenton reaction.