The incidence of DSDs has increased significantly in the last few decades, and this rise is attributed to our increased exposure to endocrine disrupting chemicals (EDCs). Prenatal exposure to the estrogenic endocrine disruptor Diethylstilbestrol (DES), a prescribed drug given to millions of pregnant women worldwide, led to multiple reproductive effects in exposed offspring. We have previously shown the effects of DES across four generations of male mice, including a significant reduction in anogenital distance, reproductive organ weights and fertility, and an increased incidence of DSDs through to the F4 generation. To determine the mechanism behind these transgenerational impacts, we investigated DNA methylation in male germ cells from three generations of DES exposed mice. Male mice were exposed to DES during gestation, and germ cells collected from control, F1, F2 and F3 DES exposed generations at day 21 post partum. Whole genome bisulfite sequencing analysis was conducted to identify differentially methylated regions (DMRs) between treatment and control germ cells. We identified 128 genes with DMRs that were common across F1, F2 and F3 generations, and had at least 20% difference in methylation compared to controls. One of the genes in which a DMR was identified was antizyme inhibitor 2 (Azin2) a modulator of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and polyamine uptake. Azin2 has an important role in testicular cells, modulating polyamine concentrations, testosterone synthesis and sperm function. In control germ cells Azin2 was 74% methylated, but this was reduced to 5% in F1, F2 and F3 generations. Methylation changes in Azin2 are therefore a potential mechanism through which DES causes transgenerational effects in exposed males. These results not only demonstrate epigenetic effects following DES exposure but also provide insights into the mechanisms through which estrogenic endocrine disruptors more broadly are impacting human fertility and reproductive health.