The present study demonstrates that 3,5,3′-tri-iodothyronine (T3) in physiological dose range inhibits tumor necrosis factor α(TNFα)/Fas-induced apoptosis in mouse hepatocytes. T3 pretreatment prevented Fas-induced early stage of apoptosis signs assessed by flow cytometry analysis of the annexin V positive cell population. T3 attenuated TNFα/Fas-induced cleavage of caspase-8 and DNA fragmentation. We found that T3 exerted its anti-apoptotic effects by mobilization of several non-genomic mechanisms independent of transcriptional activity. Inhibition of protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and Na+/H+ exchanger blocked T3-dependent anti-apoptotic effects indicating an involvement of these intracellular targets into T3-induced signaling cascade. Furthermore, physiological concentrations of T3, but not reverse T3, caused increases in intracellular cAMP content and activated PKA. T3 markedly induced phosphorylation of ERK. We also detected T3-dependent intracellular alkalinization that abolished TNFα-induced acidification. PKA inhibitor KT-5720 blocked T3-induced activation of ERK and intracellular alkalinization confirming the upstream position of PKA signaling. We further detected that hepatocytes from hypothyroid mice are more sensitive to TNFα/Fas-induced apoptosis than euthyroid animals in vivo. Together, these findings imply that T3 triggers PKA- and ERK-regulated intracellular pathways capable of driving and ensuring hepatocytes survival in the presence of death receptor ligand-induced damage under chronic inflammatory conditions.