In order to elucidate a possible role of estrogen receptor in the gonadal sex differentiation and the sex reversal with sex steroids, we examined for the formation of testis or ovary in transgenic medaka fish overexpressing the medaka estrogen receptor under the constitutive medaka β-actin promoter. The transgenic fish underwent the genetically determined gonadal differentiation and showed the same sex-reversal rates as those of wild-type non-transgenic fish after treatments with estrogen and androgen. These results present invaluable data to reconsider the role of estrogen receptor in the gonadal sex determination.