Abstract: Objective To analyze the distribution of trimethylamine N-oxide (TMAO) in healthy adults with different risk factors and explore its association with gut microbiota. Methods We collected fasting blood samples and fresh fecal samples from 181 subjects without atherogenesis in the carotid arteries. Plasma TMAO levels of the subjects were determined using stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS). The fecal DNA was extracted, and the 16S rRNA V4 tags were amplified and sequenced by Illumina HiSeq 2000. The association between TMAO and classical cardiovascular risk factors were analyzed. Gut microbial community structure was analyzed with QIIME, and LEfSe was used to identify the biomarkers. Results The median (IQR) TMAO level was 2.66 (1.96-4.91) μmol/L in the subjects. TMAO level was significantly correlated with body mass index and operational taxonomic units (OTU). Individuals with high TMAO levels were found to have abundant Clostridiales, Phascolarctobacterium, Oscillibacter, and Alistipes but less abundant Anaerosprobacter. Conclusion Chinese subjects have in general low levels of TMAO. TMAO levels are not significantly correlated with the classical cardiovascular risk factors or the gut microbial structures.