Sepsis blunts the ability of nutrient signaling by leucine to stimulate skeletal muscle protein synthesis by impairing translation initiation. The present study tested the hypothesis that overproduction of either tumor necrosis factor (TNF)-α or glucocorticoids mediate the sepsis-induced leucine resistance. Prior to producing peritonitis, rats received either vehicle, TNF binding protein (TNFBP) to inhibit endogenous TNFα action, and/or the glucocorticoid receptor antagonist RU486. Leucine was orally administered to all rats 24 h thereafter and the gastrocnemius removed 20 min later to assess protein synthesis and signaling components important in controlling peptide-chain initiation. Muscle protein synthesis was 65% lower in septic rats administered leucine than in leucine-treated control animals. This reduction was not prevented by either TNFBP or RU486 alone, but was completely reversed by the combination. This sepsis-induced leucine resistance was associated with an 80% reduction in the amount of active eIF4E·eIF4G complex, a 5-fold increase in the formation of the inactive eIF4E·4E-BP1 complex as well as markedly reduced (at least 70%) phosphorylation of 4E-BP1, eIF4G, S6K1, S6, and mTOR. Pretreatment of septic rats with either TNFBP or RU486 individually only nominally improved the leucine action as assessed by the above-mentioned endpoints. In contrast, when TNFBP and RU486 were co-administered, the ability of sepsis to impair the leucine-stimulated phosphorylation of 4E-BP1, eIF4G, S6K1, and S6 as well as the redistribution of eIF4E was essentially prevented. No differences in the total amount or phosphorylation of eIF2α and eIF2Bɛ were detected between the different groups, and changes could not be attributed to differences in the prevailing plasma concentration of insulin or leucine. Our data demonstrate the sepsis-induced leucine resistance in skeletal muscle results from the cooperative interaction of both TNFα and glucocorticoids.