Background Graves’ disease (GD) is a typical organ-specific autoimmune disease. Intestinal flora plays a pivotal role in immune homeostasis and autoimmune disease development. However, the association and mechanism between intestinal flora and GD remain elusive. Objective To investigate the association and mechanism between intestinal flora and GD. Methods We recruited 58 initially untreated GD patients and 63 healthy individuals in the study. The composition and metabolic characteristics of the intestinal flora in GD patients and the causal relationship between intestinal flora and GD pathogenesis were assessed using 16S rRNA gene sequencing, targeted/untargeted metabolomics, and fecal microbiota transplantation. Results The composition, metabolism, and inter-relationships of the intestinal flora were also changed, particularly the significantly reduced short-chain fatty acid (SCFA)-producing bacteria and SCFAs. The YCH46 strain of Bacteroides fragilis could produce propionic acid and increase Treg cell numbers while decreasing Th17 cell numbers. Transplanting the intestinal flora of GD patients significantly increased GD incidence in the GD mouse model. Additionally, there were 3 intestinal bacteria genera (Bacteroides, Alistipes, Prevotella) could distinguish GD patients from healthy individuals with 85% accuracy. Conclusions Gut dysbiosis contributes to a Treg/Th17 imbalance through the pathway regulated by propionic acid and promotes the occurrence of GD, together with other pathogenic factors. Bacteroides, Alistipes, and Prevotella have great potential to serve as adjunct markers for GD diagnosis. This study provided valuable clues for improving immune dysfunction of GD patients using B. fragilis and illuminated the prospects of microecological therapy for GD as an adjunct treatment.