AIM:To explore the correlation between trimethylamine N-oxide (TMAO) and Hashimoto's thyroiditis (HT) and to provide new ideas for early clinical diagnosis of HT. METHODS: A total of 102 patients with Hashimoto's thyroiditis (HT group) and 204 healthy individuals (control group) were included in the study and clinical data were collected. Serum TMAO levels was determined by stable isotope dilution high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). SPSS 26.0 was used for statistical analysis. RESULTS: Analysis of the baseline data revealed that there was a statistically significant difference between the TMAO levels and gender between the HT group and the control group (P<0.05). In the high-level TMAO group, the proportion of HT (63.7%) was significantly higher than that of the control group (18.6%), the regression analysis showed that high levels of TMAO were correlated with HT and positively correlated with the levels of thyroid peroxidase antibody (TPOAb), thyroglobulin antibody (TgAb), Logistic regression analysis further revealed that serum TMAO was a risk factor for the development of HT. CONCLUSION: In the TMAO>6.80 μmol/L group, the level of TMAO was correlated with HT, and the high level of TMAO was positively correlated with TPOAb and TgAb, which were risk factors for the occurrence of HT. It is suggested that TMAO can predict the risk of HT and has certain clinical value.