Atherosclerosis is accompanied by inflammation that underlies cardiovascular disease (CVD) and its vascular manifestations, including acute stroke, myocardial infarction, and peripheral artery disease, the leading causes of morbidity/mortality worldwide. The monolayer of endothelial cells formed on the luminal surface of arteries and veins regulates the vascular tone and permeability, which supports the vascular homeostasis. Endothelial dysfunction, the first step in the development of atherosclerosis, is caused by mechanical and biochemical factors which disrupt vascular home-ostasis and induce inflammation. Together with increased plasma levels of low-density lipoprotein (LDL), diabetes, hypertension, cigarette smoking, infectious microorganisms, and genetic factors, epidemiological studies established that dysregulated metabolism of homocysteine (Hcy) causing hyperhomocysteinemia (HHcy) is associated with CVD. Patients with severe HHcy exhibit severe CVD and die prematurely due to vascular complications. Biochemically, HHcy is characterized by elevated levels of Hcy and related metabolites such as Hcy-thiolactone, and N-Hcy-protein, seen in genetic and nutritional deficiencies in Hcy metabolism in humans and animals. The only known source of Hcy in humans is methionine released in the gut from dietary protein. Hcy is generated from S-adenosylhomocysteine (AdoHcy) and metabolized to cystathionine by cystathionine β-synthase (CBS) and to Hcy-thiolactone by methionyl-tRNA synthetase. Hcy-thiolactone, a chemically reactive thioester, modifies protein lysine residues generating N-homocysteinylated (N-Hcy)-protein. N-Hcy-proteins lose their normal native function, and become cytotoxic, auto-immunogenic, proinflammatory, prothrombotic, and proatherogenic. Accumulating evidence, discussed in this review, shows that these Hcy metabolites can promote endothelial dysfunction, CVD and stroke in humans by inducing pro-atherogenic changes in gene expression, upregulating mTOR signaling, and inhibiting autophagy through epigenetic mechanisms involving specific mi-croRNAs, histone demethylase PHF8, and methylated histone H4K20me1. Clinical studies, also discussed in this review, show that cystathionine and Hcy-thiolactone are associated with myo-cardial infarction and ischemic stroke by influencing blood clotting. These findings contribute to our understanding of the complex mechanisms underlying endothelial dysfunction, atherosclerosis, CVD, and stroke, and identify potential targets for therapeutic intervention.