Skeletal muscle atrophy and weakness are thought to be stimulated by tumor necrosis factor α (TNF‐α) in a variety of chronic diseases. However, little is known about the direct effects of TNF‐α on differentiated skeletal muscle cells or the signaling mechanisms involved. We have tested the effects of TNF‐α on the mouse‐derived C2C12 muscle cell line and on primary cultures from rat skeletal muscle. TNF‐α treatment of differentiated myotubes stimulated time‐ and concentration‐dependent reductions in total protein content and loss of adult myosin heavy chain (MHCf) content; these changes were evident at low TNF‐α concentrations (1–3 ng/ml) that did not alter muscle DNA content and were not associated with a decrease in MHCf synthesis. TNF‐α activated binding of nuclear factor κB (NF‐κB) to its targeted DNA sequence and stimulated degradation of I‐κBα, an NF‐κB inhibitory protein. TNF‐α stimulated total ubiquitin conjugation whereas a 26S proteasome inhibitor (MG132 10–40 μM) blocked TNF‐α activation of NF‐κB. Catalase 1 kU/ml inhibited NF‐κB activation by TNF‐α exogenous hydrogen peroxide 200 μM activated NF‐κB and stimulated I‐κBα degradation. These data demonstrate that TNF‐α directly induces skeletal muscle protein loss, that NF‐κB is rapidly activated by TNF‐α in differentiated skeletal muscle cells, and that TNF‐α/NF‐κB signaling in skeletal muscle is regulated by endogenous reactive oxygen species.—Li, Y.‐P., Schwartz, R. J., Waddell, I. D., Holloway, B. R., Reid, M. B. Skeletal muscle myocytes undergo protein loss and reactive oxygen‐mediated NF‐κB activation in response to tumor necrosis factorα. FASEB J. 12, 871–880 (1998)