DNA methylation is an epigenetic modification of cytosine residues within CpG dinucleotides at about 20-30 million sites in the mammalian genome and is involved in gene regulation, X inactivation and genomic imprinting (variation in the expression of a subset of genes according to their maternal or paternal origin). Abnormalities in DNA methylation have been associated with perturbations in growth, placental function, neurobehavioral processes, and carcinogenesis. Methylation of DNA is catalyzed by DNA (cytosine-5)-methyltransferases (DNMTs) and is acquired in a sex-specific manner in the germline. In the male germline, the bulk of gamete methylation is initially acquired during germ cell development in the fetal testis; DNA methylation pattern acquisition is then completed postnatally during the transition from spermatogonia to pachytene spermatocytes. Data from mouse and human studies indicate that genome-wide methylation patterns are unique in sperm as compared to the patterns in somatic tissues. Cellular expression studies and gene-targeting experiments have identified critical roles for DNMT3a and DNMT3L in establishing methylation patterns on imprinted as well as single-copy, repeat and intergenic sequences during male germ cell development. Methyl donor (e.g. folate) supply to developing germ cells is essential during the prenatal and postnatal phases of DNA methylation acquisition. Deficiency in the key folate pathway enzyme methylenetetrahydrofolate reductase (MTHFR) is associated with DNA methylation defects in sperm and infertility that can be partially alleviated by provision of dietary methyl donors.