<p>Folate is an essential nutrient that supports the formation of S-adenosyl methionine (SAM) in the pathway of one carbon metabolism. Dietary folate intake has been shown to affect the SAM-dependent methylation of diverse substrates, including DNA and inorganic arsenic (iAs). The methylation of iAs by arsenic methyltransferase (AS3MT) plays a key role iAs detoxification in both humans and mice. Our recent studies using wild-type C57BL/6N mice showed that preconception exposure to iAs resulted in heritable changes in DNA methylation in paternal sperm and differential expression of genes in tissues of the offspring that developed a diabetic phenotype. The goal of the present study was to determine if dietary folate can modify the iAs-induced differential methylation of DNA in sperm of male C57BL/6 mice expressing the human AS3MT and exhibiting a human-like pattern of iAs metabolism. Mice were fed folate deficient (FD, 0&#xa0;mg folic acid/kg) or folate supplemented (FS, 10&#xa0;mg folic acid/kg) diet for 6 weeks, followed by exposure to 0 (controls) or 400 ppb iAs (arsenite) in drinking water for 5 weeks while on the same types of diet. Reduced Representation Bisulfite Sequencing was used to identify CpG sites and genes that were differentially methylated in response to iAs exposure, followed by analysis of pathways enriched for these genes. Genes and pathways associated with cell morphology and function, and neural structure and function were the top pathways enriched by iAs exposure in both FD and FS mice. Notably, pathways associated with diabetes, regulation of insulin secretion and signaling were enriched for differentially methylated genes only in the sperm of iAs-exposed FS mice. These results suggest that folate intake modifies the effects of iAs exposure on DNA methylation in sperm of the humanized mice, providing strong rationale for studies that will examine the role of folate in modulation of adverse effects of preconception exposure to iAs.</p>