Di (2-ethylhexyl) phthalate (DEHP), an estrogen-like compound that is a ubiquitous environmental contaminant, has been reported to adversely affect human and mammalian reproduction. Many studies have found that exposure to DEHP during pregnancy perturbs female germ cell meiosis and is detrimental to oogenesis. Previous studies have demonstrated that melatonin (MLT) is beneficial to reproductive endocrinology, oogenesis, and embryonic development as the ability to antioxidative and antiapoptotic. However, whether the meiotic defect of germ cells exposed to DEHP could be rescued by MLT is not clear. Here, we cultured 12.5 days post coitum (dpc) fetal mouse ovaries for 6 days, exposed them to 100 μM DEHP with or without 1 μM MLT in vitro.. The results showed that DEHP exposure induced the abnormal formation of DNA double-strand breaks (DSBs), and inhibited the repair of DSBs during meiotic recombination. In addition, we found defective oocytes were prone to undergo apoptosis. Notably, this defect could be remarkably ameliorated by the addition of MLT via a reduction of the levels of reactive oxygen species and an inhibition of apoptosis. In conclusion, our data revealed that MLT had a protective action against the meiotic deterioration of fetal oocytes induced by DEHP in the mouse in vitro.