<p><b>BACKGROUND: </b>Trimethylamine-N-oxide (TMAO), a diet-derived and gut microbiota-related metabolite, is associated with cardiovascular disease (CVD). However, major dietary determinants and specific gut bacterial taxa related to TMAO remain to be identified in humans.</p><p><b>OBJECTIVES: </b>We aimed to identify dietary and gut microbial factors associated with circulating TMAO.</p><p><b>METHODS: </b>This cross-sectional study included 3972 participants (57.3% women) aged 18-74 y from the Hispanic Community Health Study/Study of Latinos in the United States. Dietary information was collected by 24-h dietary recalls at baseline interview (2008-2011), and baseline serum TMAO and its precursors were measured by an untargeted approach. Gut microbiome was profiled by shotgun metagenomic sequencing in a subset of participants (n = 626) during a follow-up visit (2016-2018). Logistic and linear regression were used to examine associations of inverse-normalized metabolites with prevalent CVD, dietary intake, and bacterial species, respectively, after adjustment for sociodemographic, behavioral, and clinical factors.</p><p><b>RESULTS: </b>TMAO was positively associated with prevalent CVD (case number = 279; OR = 1.34; 95% CI: 1.17, 1.54, per 1-SD). Fish (P = 1.26 × 10-17), red meat (P = 3.33 × 10-16), and egg (P = 3.89 × 10-5) intakes were top dietary factors positively associated with TMAO. We identified 9 gut bacterial species significantly associated with TMAO (false discovery rate <0.05). All 4 species positively associated with TMAO belong to the order Clostridiales, of which 3 might have homologous genes encoding carnitine monooxygenase, an enzyme converting carnitine to trimethylamine (TMA). The red meat-TMAO association was more pronounced in participants with higher abundances of these 4 species compared with those with lower abundance (Pinteraction = 0.013), but such microbial modification was not observed for fish-TMAO or egg-TMAO associations.</p><p><b>CONCLUSION: </b>In US Hispanics/Latinos, fish, red meat, and egg intakes are major dietary factors associated with serum TMAO. The identified potential TMA-producing gut microbiota and microbial modification on the red meat-TMAO association support microbial TMA production from dietary carnitine, whereas the fish-TMAO association is independent of gut microbiota.</p>