The neutrophil-derived serine protease, cathepsin G (Cat.G), has been shown to induce myocyte detachment and apoptosis by anoikis through down-regulation of focal adhesion (FA) signaling. However, the mechanisms that control FA protein stability and turnover in myocytes are not well understood. Here, we have shown that the Casitas b-lineage lymphoma (c-Cbl), adaptor protein with an intrinsic E3 ubiquitin ligase activity, is involved in FA and myofibrillar protein stability and turnover in myocytes. Cat.G treatment induced c-Cbl activation and its interaction with FA proteins. Deletion of c-Cbl using c-Cbl knock-out derived myocytes or inhibition of c-Cbl ligase activity significantly reduced FA protein degradation, myofibrillar degeneration, and myocyte apoptosis induced by Cat.G. We also found that inhibition of the proteasome activity, but not the lysosome or the calpain activity, markedly attenuated FA and myofibrillar protein degradation induced by Cat.G. Interestingly, c-Cbl activation induced by Cat.G was mediated through epidermal growth factor receptor (EGFR) transactivation as inhibition of EGFR kinase activity markedly attenuated c-Cbl phosphorylation and FA protein degradation induced by Cat.G. These findings support a model in which neutrophil protease Cat.G promotes c-Cbl interaction with FA proteins, resulting in enhanced c-Cbl-mediated FA protein ubiquitination and degradation, myofibril degradation, and subsequent down-regulation of myocyte survival signaling.