During the long migration from river habitats to the spawning ground, the Japanese eel undergoes sexual maturation. This spawning migration occurs concurrently with morphological changes, such as increases in eye size; however, the mechanisms by which sex steroids and their receptors influence these changes in peripheral tissues remain unclear. The aim of this study was to investigate changes in the eyes of female Japanese eels during sexual maturation, and our research focused on estrogen receptor (ER)α and ERβ transcripts. During ovarian development, the gonadosomatic index increased and yolk-laden oocytes developed rapidly. These changes occurred in conjunction with a steady increase in plasma levels of estradiol-17β (E₂). Concomitant increases in transcript levels of ERα and ERβ in eye, brain, pituitary, and ovary were also observed. Fluorescence in-situ hybridization analyses revealed that ERα and ERβ transcripts were present in the choriocapillary layer and photoreceptor layer of the eyes, and the analysis also revealed that their signals in these layers became stronger in mature females compared to those observed in immature females, suggesting that under the influence of gonadotropins, morphological changes in the eyes are regulated by E₂ through the activation of its receptors. In conclusion, E₂ plays a crucial role in physiological adaptations that occur in peripheral tissues during the spawning migration.