Estrogen, which is synthesized earlier in females than androgen in males, is critical for sex determination in non-mammalian vertebrates. However, it remains unknown that what would happen to the gonadal phenotype if estrogen and androgen were administrated simultaneously. In this study, XY and XX tilapia fry were treated with the same dose of 17a-methyltestosterone (MT) and 17b-estradiol (E 2 ) alone and in combination from 0 to 30 days after hatching. Treatment of XY fish with E 2 resulted in male to female sex reversal, while treatment of XX fish with MT resulted in female to male sex reversal. In contrast, simultaneous treatment of XX and XY fish with MT and E 2 resulted in female, but with cyp11b2 and cyp19a1a co-expressed in the ovary. Serum 11-ketotestosteron level of the MT and E 2 simultaneously treated XX and XY female was similar to that of the XY control, while serum E 2 level of these two groups was similar to that of the XX control. Transcriptomic cluster analysis revealed that the MT and E 2 treated XX and XY gonads clustered into the same branch with the XX control. However a small fraction of genes, which showed disordered expression, may be associated with stress response. These results demonstrated that estrogen could maintain the female phenotype of XX fish and feminize XY fish even in the presence of androgen. Simultaneous treatment with estrogen and androgen up-regulated the endogenous estrogen and androgen synthesis, and resulted in disordered gene expression and endocrine disruption in tilapia.