BACKGROUND: Environmental endocrine disruptor Di (2-ethylhexyl) phthalate (DEHP) widely affects the health of human and animals including the reproductive system. However, there are few studies on the protective strategies for the maternal DEHP exposure on follicular development of offspring. In the present study, we established a model of lactation female mice exposed to DEHP and reported the effects and potential mechanism of melatonin on the follicular development of offspring. RESULTS: Our data showed that melatonin rescued the decrease of primordial follicles, antral follicles and oocyte number (increased by 74.2%) of offspring caused by maternal DEHP exposure from the primordial follicle formation stage. Proteomic analysis showed that melatonin altered the ovarian steroidogenesis, lipid metabolism, signal transduction, and DNA damage-related proteins. Melatonin reversed the disorder of lipid metabolism caused by DEHP and stabilized ovarian hormone secretase level. Molecular docking results indicated that DEHP/MEHP/melatonin binds to HSD17B2 to form a stable conformation, which may explain the reduction in 17β-estradiol induced by DEHP. Moreover, melatonin restored granulosa cell proliferation, reduced oxidative stress and DNA damage-related apoptosis, enhanced mitochondrial function, and protected ovarian cells. Besides, melatonin enhanced gap junction and promoted intercellular communication, which facilitate the formation of primordial follicles and the growth and development of antral follicles. In addition, melatonin rescued the oocyte defects of offspring caused by maternal DEHP exposure. CONCLUSIONS: Taken together, our data showed that melatonin could alleviate the damage of follicular development and abnormal ovarian steroidogenesis of offspring caused by maternal DEHP exposure.