Vertebrate sex development consists largely of two processes: "sex determination，" the initial bifurcation of sexual identity， and "sex differentiation，" which subsequently facilitates maleness or femaleness according to the sex determination signal. Steroid hormones promote multiple types of sexual dimorphism in eutherian mammals and avians [1-3]， in which they are indispensable for proper sex differentiation. By contrast， in many poikilothermic vertebrates， steroid hormones have been proposed to be key players in sex determination as well as sex differentiation [4-8]. This hypothesis was introduced more than 50 years ago but has never been rigorously tested due to difficulties in discriminating the roles of steroids in sex determination and differentiation. We found that a missense SNP in the gene encoding the steroidogenic enzyme 17β-hydroxysteroid dehydrogenase 1 (Hsd17b1) was perfectly associated with ZZ/ZW sex determination in Seriola fishes. Biochemical analyses revealed that a glutamate residue present specifically in Z-type HSD17B1 attenuated interconversion between 17-keto and 17β-hydroxy steroids relative to the allelic product from the W chromosome， which harbors glycine at that position， by disrupting the hydrogen bond network between the steroid and the enzyme's catalytic residues. Hsd17b1 mRNA is constitutively expressed in undifferentiated and differentiating gonads of both genotypic sexes， whereas W-type mRNA is expressed only in genotypic females. Meanwhile， Cyp19a1 is?predominantly expressed in differentiating ovary. We conclude that the combination of Hsd17b1 alleles determines sex by modulating endogenous estrogen levels in Seriola species. These findings strongly support the long-standing hypothesis on steroids in sex determination.