The aim was to study the effect of 17α‐ethynylestradiol (EE2) on osmoregulation of fish, adult zebrafish (Danio rerio) were exposed to 10 ng/L and 100 ng/L of EE2 for 7 days. After exposure, EE2 induced body oedema, trichangiectasis in the skin and protrusion of the proptosis and significantly elevated the condition factor, water weight and water content, decreased the RBC count and HCT of the fish. In addition, various histopathological damages in the gill and kidney were observed. We therefore speculate that EE2 exposure may disrupt water homeostasis in zebrafish. Further research found that the mRNA levels of crh, crhr and acth were remarkably down‐regulated in the brain, in accordance with the decreased level of plasma cortisol. Moreover, remarkably increased aqp3a and aqp1a1 mRNA levels in the gill were observed, suggesting elevated water intake in the gill. Meanwhile, decreased expression of atp1a1a.1 and the activity of Na+, K+‐ATPase in the gill, which might have resulted from lower plasma cortisol levels, were also present, in consistence with the lesions of histocyte. Moreover, suppressed expression of atp1a1a.4 and the activity of Na+, K+‐ATPase in the kidney were probably resulted from decreased plasma cortisol levels and low expression of avp in the brain.