Vascular oxidative stress serves as a pathological foundation for various vascular injury-related diseases, including atherosclerosis, hypertension, restenosis, and abdominal aortic aneurysms. Recent studies have indicated that intestinal flora-derived metabolites, especially phenylacetylglutamine (PAGln) and phenylacetylglycine (PAGly), may contribute to the promotion of thrombosis, heart failure, and other related conditions. Aucubin (AU), an iridoid glycoside, has been shown to exhibit anti-cardiovascular properties. Nevertheless, the precise role and underlying mechanisms by which AU mitigates PAGly-induced vascular injury remain poorly understood. Our results indicated that PAGln/PAGly promoted oxidative stress in vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in vitro and in vivo. Network pharmacology suggest that AU may possess the capacity to regulate lipid and atherosclerosis, and reactive oxygen species (ROS) processes. We found that AU penetrated the blood vessels and mitigated oxidative stress induced by PAGln/PAGly. Mechanistically, combining the results from intersection analysis between the targets of AU and vascular diseases and molecular docking, we found that tumor necrosis factor (TNF) may be the potential target of AU. Further DARTS and molecular docking analysis demonstrated that AU bound to recombinant TNF-α, and AU could interact with multiple amino acid residues of TNF-α, including Asn-92 and Phe-144. Additionally, PAGly upregulated the level of soluble TNF-α (sTNF-α) in mouse VSMCs and plasma, and promoted the interaction between sTNF-α and TNF receptor 1 (TNFR1), whereas AU inhibited this interaction. Both AU and Infliximab, a specific monoclonal antibody of TNF-α, inhibit TNF-α-induced ROS production. In summary, our results revealed that TNF-α is a cellular target of AU, and the interaction between AU and sTNF-α may mitigate PAGln/PAGly-induced vascular oxidative stress by inhibiting the interaction of TNF-α-TNFR1. Keywords