Trimethylamine N-oxide (TMAO) is a circulating metabolite that has been implicated in the development of atherosclerosis and cardiovascular disease (CVD). In this paper, we identify blood markers, metabolites, proteins, gut microbiota patterns, and diets that are significantly associated with levels of plasma TMAO. We find that kidney markers are strongly associated with TMAO and identify CVD-related proteins that are positively correlated with TMAO. We show that metabolites derived by the gut microbiota are strongly correlated with TMAO and that the magnitude of this correlation varies with kidney function. Moreover, we identify diet-associated patterns in the microbiome that are correlated with TMAO. These findings suggest that both the process of TMAO accumulation and the mechanism by which TMAO promotes atherosclerosis are a complex interplay between diet and the microbiome on one hand and other system-level factors such as circulating proteins, metabolites, and kidney function. Display omitted •TMAO is independently associated with demographic factors and kidney function•Gut microbiota-derived metabolites are significantly associated with TMAO•Diet- and disease-associated metabolites are significantly associated with TMAO•Proteins linked to cardiovascular and kidney disease are correlated with TMAO Trimethylamine N-oxide (TMAO) is a circulating metabolite that has been implicated in the development of atherosclerosis and cardiovascular disease. Manor et al. identify blood markers, metabolites, proteins, gut microbiota patterns, and diets that are significantly associated with levels of plasma TMAO.