Evidence from previous studies suggests that the male reproductive system can be disrupted by fetal or neonatal exposure to diethylstilbestrol (DES). However, the molecular basis for this effect remains unclear. To evaluate the effects of DES on mouse spermatocytes and to explore its potential mechanism of action, the levels of DNA methyltransferases (DNMTs) and DNA methylation induced by DES were detected. The results showed that low doses of DES inhibited cell proliferation and cell cycle progression and induced apoptosis in GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line, which reproduces primary cells responses to E2. Furthermore, global DNA methylation levels were increased and the expression levels of DNMTs were altered in DES-treated GC-2 cells. A total of 141 differentially methylated DNA sites were detected by microarray analysis. Rxra, an important component of the retinoic acid signaling pathway, and mybph, a RhoA pathway-related protein, were found to be hypermethylated, and Prkcd, an apoptosis-related protein, was hypomethylated. These results showed that low-dose DES was toxic to spermatocytes and that DNMT expression and DNA methylation were altered in DES-exposed cells. Taken together, these data demonstrate that DNA methylation likely plays an important role in mediating DES-induced spermatocyte toxicity in vitro.