<p>As a potent thyroid hormone, triiodothyronine (T₃) exhibits numerous physiological actions in fish, including regulation of ion and osmotic balance. It targets the liver and plays an essential role in the growth and metabolism of fish. However, it is uncertain whether T₃ directly affects ion osmotic regulation and mitochondrial energetics in hepatic tissue, particularly in stressed fish. We, therefore, tested the dose-responsive in vitro action of T₃ [10⁻⁹&#xa0;M (low), 10^–8 (mid) and 10^–7 (high) doses], on the ion osmotic transporter function and mitochondrial energetic markers in hepatic explants of air-breathing fish <i>Anabas testudineus</i> to delineate the rapid in vitro action of T₃ in stressed fish liver. We found that in vitro T₃ increased hepatic nitric oxide (NO) content, but lowered the hepatic reactive oxygen species status in non-stressed fish. Likewise, in non-stressed fish liver, the low dose of T₃ lowered, and the middle dose elevated cytochrome c oxidase activity. T₃ increased lactate dehydrogenase activity but decreased succinate dehydrogenase activity. T₃ exposure modified the transport of Na⁺, K⁺ and H⁺, NH₄⁺ ions, as evident from the altered Na⁺/K⁺ ATPase Na⁺/NH4⁺, H⁺/K⁺ ATPase and vacuolar H⁺ ATPase activities in the hepatic explants. Likewise, T₃ rapidly altered the Ca²⁺, Mg²⁺ and H⁺ ion-dependent ATPase transport activities in the hepatic mitochondria. Interestingly, a reversed response pattern of NO status, energetic markers and ion transporter activities was found after T₃ exposure in the stressed fish hepatic explants. The data indicate a rapid action of T₃ on the performance and intensity of the energy-driven ion osmotic stress response in this vital tissue. Overall, the data support the hypothesis that T₃ rapidly influences energetic and ion osmotic responses in stressed hepatic tissue, offering protection against stress and thereby confirming the critical modifying role of T₃ in the hepatic stress acclimation of fish.</p>