In the present study, we evaluated the changes in gonad-somatic index (GSI), as well as stress and reproductive-related gene expression in female zebrafish (Danio rerio) exposed to environmentally relevant and pharmacological concentrations of 17alpha-ethinylestradiol (EE2; 0.5-500 ng/L) for 21 days. Although the endocrine-disrupting effects of EE2 are well-documented, there remains a gap in understanding its concurrent impact on both reproductive and stress axes, particularly at pharmacological doses. Therefore, this study aimed to fill this gap by simultaneously assessing steroidogenesis, stress responses, and their molecular regulators. Fish mortality was significantly higher at the two highest concentrations (50 and 500 ng EE2/L), while no mortality occurred at lower concentrations. A significant increase in whole-body vitellogenin (Vtg) and mRNA level of Vtg1 was observed at concentrations of 5 ng/L and above, with no significant differences among these groups. In contrast, the levels of sex steroids (17beta-estradiol and testosterone), as well as the expression of reproductive-related genes (StAR, cyp19a1), significantly decreased at 50 and 500 ng/L. Additionally, cortisol levels and glucocorticoid receptor (GR) gene expression were significantly reduced at these same concentrations. In conclusion, this study provides novel evidence that EE2, particularly at pharmacological doses of 50 and 500 ng/L, simultaneously disrupts steroidogenesis and impairs the stress axis in female zebrafish, likely through interference with the expression of key genes governing sex steroid synthesis and stress regulation.