This study investigates the role of gut flora in the metabolism of phosphatidylcholine (PC) and its impact on cardiovascular disease (CVD). Metabolomics analysis identified three metabolites of PC—choline, trimethylamine N-oxide (TMAO), and betaine—as predictors of CVD risk. Dietary supplementation of mice with choline, TMAO, or betaine increased macrophage scavenger receptors linked to atherosclerosis and promoted atherosclerosis. Germ-free mice confirmed the critical role of dietary choline and gut flora in TMAO production and macrophage cholesterol accumulation. Genetic variations controlling expression of flavin monooxygenases (FMOs), which are enzymes that produce TMAO, were associated with atherosclerosis in hyperlipidemic mice. The study suggests that gut flora-dependent metabolism of dietary PC and its metabolites may contribute to CVD pathogenesis, providing opportunities for novel diagnostic tests and therapeutic approaches for atherosclerotic heart disease.This study investigates the role of gut flora in the metabolism of phosphatidylcholine (PC) and its impact on cardiovascular disease (CVD). Metabolomics analysis identified three metabolites of PC—choline, trimethylamine N-oxide (TMAO), and betaine—as predictors of CVD risk. Dietary supplementation of mice with choline, TMAO, or betaine increased macrophage scavenger receptors linked to atherosclerosis and promoted atherosclerosis. Germ-free mice confirmed the critical role of dietary choline and gut flora in TMAO production and macrophage cholesterol accumulation. Genetic variations controlling expression of flavin monooxygenases (FMOs), which are enzymes that produce TMAO, were associated with atherosclerosis in hyperlipidemic mice. The study suggests that gut flora-dependent metabolism of dietary PC and its metabolites may contribute to CVD pathogenesis, providing opportunities for novel diagnostic tests and therapeutic approaches for atherosclerotic heart disease.