The gastrointestinal degradation of choline, betaine, and carnitine results in the formation of trimethylamine N-oxide (TMAO) in humans and dairy cows. In humans, TMAO has emerged as an associative biomarker for non-alcoholic fatty liver disease, cardiovascular disease, and type 2 diabetes; however, conflicting data is emerging. This presentation will explore the role of TMAO, the effect of diet on the gut microbiome in relation to TMAO formation, and new work focused on the effects or lack thereof in dairy cows.