Freshwater (FW)-adapted tilapia (Oreochromis mossambicus) were treated with estradiol (E₂) for 4 days to stimulate protein synthesis and sampled at 0, 4, and 24 h after exposure to 50% seawater (SW). E₂ increased circulating vitellogenin (VTG) levels in large amounts, indicative of unusually high rates of hepatic protein synthesis. E₂ treatment prevented the recovery of plasma osmolality in 50% SW that was evident in the sham group. Plasma sodium concentration was significantly elevated with E₂ in FW, but the levels did not change in 50% SW. Gill Na⁺-K⁺-ATPase activity was significantly lower in the E₂ group compared with sham-injected tilapia in 50% SW. No significant differences were noted in plasma cortisol, thyroxine, triiodothyronine, or glucose concentration with E₂ in 50% SW. E₂ significantly lowered several key liver enzyme activities and also decreased gill lactate dehydrogenase and malate dehydrogenase activities over a 24-h period. Together, our results suggest that E₂ impairs ion regulation in tilapia, partially mediated by a decreased metabolic capacity in liver and gill. The decreased tissue metabolic capacity is likely due to E₂-induced energy repartitioning processes that are geared toward VTG synthesis at the expense of other energy-demanding pathways.