Graves' orbitopathy (GO) is a sight-threatening organ-specific autoimmune disease with complicated pathogenesis. Gut microbiota-derived tryptophan (Trp) metabolites play important roles in immune-related diseases, but their role in GO remains unknown.Trp metabolism-associated gut flora was analyzed by 16 S sequencing in GO patients and controls. Serum metabolomics profiling was performed to assess Trp metabolic pathway. Trp metabolites levels were measured by ELISA in 401 serum samples from a case-control study, and their effects on inflammation and proliferation in orbital fibroblasts were evaluated in vitro.Trp metabolism-associated gut flora, including phylum Firmicutes and genus Anaerostipes, were significantly down-regulated in GO patients. Serum metabolomics revealed significant enrichment of Trp metabolic pathway in both GO and Graves' disease (GD) groups. Serum levels of indolepropionic acid (IPA), indole-3-lactate (ILA), and indoleacetic acid (IAA) were significantly decreased in both GD and GO patients compared to controls, with IAA levels further reduced in GO compared to GD patients. Notably, active GO patients had significantly lower IAA levels compared to inactive ones. Moreover, the levels of IAA were negatively correlated with clinical activity score and serum thyrotropin receptor antibody (TRAb) in GO patients. In vitro, IPA, ILA, and IAA mitigated TNFα-induced inflammation and proliferation in orbital fibroblasts by suppressing the Akt signaling pathway.Trp metabolites IAA maybe a novel biomarker for GO progression. And IPA, ILA and IAA may play a protective role in GO by regulating inflammation and proliferation in orbital fibroblasts, suggesting their potential as therapeutic targets for GO treatment.© 2025. The Author(s).