Background &amp; Aims <p>Dysfunction of the intestinal epithelial barrier (IEB) and gut vascular barrier (GVB) contributes to the development of intestinal ischemia/reperfusion (IR) injury. Tryptophan (TRP), an essential amino acid, plays a crucial role in maintaining intestinal homeostasis, yet its regulatory effects on the GVB following IR remain unexplored. We aimed to better define the role of TRP in intestinal IR in vivo and in vitro.</p> Methods <p>Mice underwent intestinal ischemia/reperfusion (IR) and were fed control, TRP-recommended (TRP-r), or TRP-sufficient (TRP-s) diets. Fecal metagenomic sequencing analyzed microbial composition, and targeted metabolomics quantified tryptophan and its metabolites in intestinal and serum samples. ILA’s effects on barrier integrity were assessed via tight junction protein expression and FITC-dextran permeability assays. RNA sequencing of intestinal endothelial cells elucidated mechanisms by which ILA modulated GVB function. The STAT3-claudin 2 relationship was validated in vitro by ChIP-qPCR.</p> Results <p>TRP supplementation significantly reshaped the gut microbiota, mitigated tissue damage and enhanced the integrity of both the IEB and GVB. Indole-3-lactic acid (ILA), a key tryptophan metabolite, was identified as an important factor in preserving GVB function. Mechanistically, our results show that the aryl hydrocarbon receptor (AhR)/Nrf2/signal transducer and activator of transcription 3 (STAT3) pathway is essential for ILA-mediated improvement of GVB integrity and downregulation of the pore-forming protein claudin 2.</p> Conclusions <p>Our findings highlight the dual role of ILA in reinforcing both IEB and GVB functions and shed light on the molecular mechanisms underlying ILA’s GVB-protective effects. This study implicates that ILA or other AhR-activating metabolites may serve as promising pharmacological agents for alleviating IR-induced intestinal damage.</p> Graphical Abstract <p></p>