Disruption in bile duct barrier function contributes to hepatocyte toxicity in ischemia-reperfusion injury, often leading to surgical complications in liver resection, transplantation, and hemorrhagic shock. However, the underlying mechanisms remain incompletely understood.Transcriptomic and proteomic analyses were conducted to examine tryptophan (Trp) metabolism in a Pringle maneuver-induced bile duct injury rat model; Hypoxia/Reoxygenation (H/R) was used to establish an in vitro cholangiocyte injury model. Cholangiocyte injury was assessed via hematoxylin and eosin (H&E) staining, Ki67/myeloperoxidase (MPO) immunohistochemistry, transmission electron microscopy (TEM), and TUNEL/CK19 co-staining. Tight junction integrity was evaluated by measuring transepithelial electrical resistance (TEER), inulin permeability, and confocal immunofluorescence (IF) for ZO-1/CK19 co-staining. Gene expression was quantified using RT-qPCR and Western blotting, while metabolites were analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS).Significant alterations in Trp metabolism-related genes (Kynu, Haao, Kat1/Kat2) and metabolites were observed. Continuous Pringle maneuver resulted in elevated levels of 3-hydroxyanthranilic acid (3-HAA) and quinolinic acid (QA), a decreased xanthurenic acid (XA) level. In vitro, Kynu inhibition, using shRNA or the inhibitor benserazide (BSZ), ameliorated tight junction impairment and attenuated inflammatory damage in hypoxic biliary epithelial cells. In vivo, post-ischemia Kynu blockade reduced bile duct damage, inflammation, and biliary barrier permeability. Proteome analysis revealed that Kynu inhibition decreased 3-HAA, AA and QA levels while increased XA level. Notably, XA (but not AA or QA) treatment restored cell junction integrity under hypoxic conditions and modulated cytokine expression, potentially via ZO1 regulation through the GluR2/CX50 pathway. By day 7, BSZ or XA administration reduced serum bilirubin levels and mitigated of bile duct hyperplasia.Our findings demonstrated that Kynu inhibition alleviates bile duct ischemic injury by reprogramming dysregulated tryptophan metabolism, particularly through XA upregulation. This modulation may restore tight junction function via the GluR2/3/CX50-ZO1 axis, thereby preserving blood-biliary barrier integrity. Targeting Kynu represents a promising therapeutic strategy for ischemia-induced bile duct injury.© 2025. The Author(s).