Nuclear factor-κB (NF-κB) regulates the transcription of a variety of genes involved in immune responses, cell growth, and cell death. However, the role of NF-κB in muscle biology is poorly understood. We recently reported that tumor necrosis factor-α (TNF-α) rapidly activates NF-κB in differentiated skeletal muscle myotubes and that TNF-α acts directly on the muscle cell to induce protein degradation. In the present study, we ask whether NF-κB mediates the protein loss induced by TNF-α. We addressed this problem by creating stable, transdominant negative muscle cell lines. C2C12 myoblasts were transfected with viral plasmid constructs that induce overexpression of mutant I-κBα proteins that are insensitive to degradation via the ubiquitin-proteasome pathway. These mutant proteins selectively inhibit NF-κB activation. We found that differentiated myotubes transfected with the empty viral vector (controls) underwent a drop in total protein content and in fast-type myosin heavy-chain content during 72 h of exposure to TNF-α. In contrast, total protein and fast-type myosin heavy-chain levels were unaltered by TNF-α in the transdominant negative cell lines. TNF-α did not induce apoptosis in any cell line, as assessed by DNA ladder and annexin V assays. These data indicate that NF-κB is an essential mediator of TNF-α-induced catabolism in differentiated muscle cells.