Technical Abstract: The regulation of site-specific DNA methylation of tumor suppressor genes has been considered as a leading mechanism by which certain nutrients exert their anticancer property. Our previous studies suggest that dietary selenium (Se) may alter DNA methylation, and the purpose of this study was to investigate whether long term consumption of dietary Se affects the methylation of genomic DNA, exon-specific p53 DNA gene. Three groups of rats (n =6-7 /group) were fed the AIN-93G basal diet supplemented with 0 (Se deficient), 0.15 (Se adequate), or 4 mg (Se supranutritional) (Se as L-selenomethionine)/kg diet for 104 days, respectively. Rats fed Se deficient or supranutritional diet had greater plasma and liver glutathione peroxidase activity, liver thioredoxin reductase activity and plasma homocysteine level than those without Se supplementation. However, compared with Se adequacy, Se supranutrion did not further increase these Se dependent enzyme activities and homocysteine level. In contrast, Se contents in kidney, liver, gastrocnemus muscle and plasma were increased in a Se-dose dependent manner. Interestingly, rats fed Se supranutritional diet significantly decreased global genomic DNA methylation in rat liver. However, Se supranutritional diet greatly increased the methylation of p53 gene (exons 5–8) but not B-actin gene (exon 2-3) DNA in liver and colon mucosa. Taken together, the long term Se consumption not only affects Se contained enzyme activities, homocysteine level and tissue Se contents but also produces an opposing effect on the methylation of globe genomic DNA and tumor suppressor p53 gene.