Hyperhomocysteinemia is a risk factor for atherosclerosis and vascular disease; however, the mechanism underlying this association remains poorly understood. Increased levels of intracellular S-adenosylhomocysteine (AdoHcy), secondary to homocysteine-mediated reversal of the AdoHcy hydrolase reaction, have been associated with reduced DNA methylation patterns and pointed as responsible for the hyperhomocysteinemia-related endothelial dysfunction. Methylation is an epigenetic feature of genomic DNA, which leads to alterations in gene expression. So far, the effect of intracellular AdoHcy accumulation on DNA methylation patterns has not yet been fully substantiated by experimental evidence. The present study was designed to evaluate, in cultured endothelial cells, the effect of AdoHcy accumulation on genomic global DNA methylation status. Experimental intracellular accumulation of AdoHcy was induced by adenosine-2,3-dialdehyde (ADA), an inhibitor of AdoHcy hydrolase. Increased concentrations of inhibitor were tested, and unsupplemented medium incubations were used as controls. Cytosolic and nuclear fractions were obtained from trypsinized cells after 72 h of incubation. Total homocysteine concentration was quantified (culture medium and cytosolic fractions) by high-performance liquid chromatography (HPLC). S-Adenosylmethionine and AdoHcy concentrations were measured (cytosolic fractions) by stable-isotope dilution LC-tandem mass spectrometry method. Genomic DNA was obtained from the nuclear fraction, and global DNA methylation status was evaluated by the cytosine extension assay. The results showed that supplementation of the culture medium with ADA had no cytotoxic effect and increased the intracellular AdoHcy concentration in a dose-dependent manner. A significant negative correlation was observed between intracellular AdoHcy and genomic DNA methylation status. These findings strongly point to the importance of AdoHcy as a pivotal biomarker of genomic DNA methylation status.