The follicle is an important unit for the synthesis of steroid hormones and the oocyte development and maturation in mammals. However, the effect of methionine supply on follicle development and its regulatory mechanism are still unclear. In the present study, we found that dietary methionine supplementation during the estrous cycle significantly increased the number of embryo implantation sites, as well as serum contents of a variety of amino acids and methionine metabolic enzymes in rats. Additionally, methionine supplementation markedly enhanced the expression of rat ovarian neutral amino acid transporters, DNA methyltransferases (DNMTs), and cystathionine gamma-lyase (CSE); meanwhile, it significantly increased the ovarian concentrations of the metabolite S-adenosylmethionine (SAM) and glutathione (GSH). In vitro data showed that methionine supply promotes rat follicle development through enhancing the expression of critical gene growth differentiation factor 9 and bone morphogenetic protein 15. Furthermore, methionine enhanced the relative protein and mRNA expression of critical genes related to estrogen synthesis, ultimately increasing estrogen synthesis in primary ovarian granulosa cells. Taken together, our results suggested that methionine promoted follicular growth and estrogen synthesis in rats during the estrus cycle, which improved embryo implantation during early pregnancy. These findings provided a potential nutritional strategy to improve the reproductive performance of animals.