In order to elucidate the role of estrogen in the female sex differentiation of Takifugu obscurus, partial sequences of CYP19A and DMRT1 cDNA in gonad of T.obscurus were cloned respectively. T.obscurus flies were exposed to 0, 25, 125, 625, 3 125 μg/L aromatase inhibitor letrozole (LE) to investigates the CYP19A and DMRT1 gene expression and histological changes. RT-PCR results showed that the CYP19A andDMRT1 expression level in 25 μg/L LE group did not differ from that of the control. In about 30% of the samples exposed to 125 μg/L or higher LE, both CYP19A and DMRT1 genes were expressed. With the increase of LE concentration, the CYP19A expression level decreased, while the DMRT1 expression level increased. Histologically, the samples treated with 125 μg/L or higher LE exhibited visible intersex gonads during their sex reversal from female to male. Fifty-six days after hatching, about 20% of the samples treated with 125 μg/L LE showed phenotypic female; the samples treated with 625 μg/L LE showed phenotypic male and intersex, without phenotypic female; all samples treated with 3 125 μg/L LE showed phenotypic male, and their CYP19A and DMRT1 expression levels were consistent with those of the control males. Conclusion: Inhibition of endogenous estrogen synthesis allows CYP19A expression downregulation, and DMRT1 expression upregulation inT.obscurus fly, and the occurrence of female-to-male reversal.