Examination of the antioxidative homeostasis in skeletal muscle cells in the presence or absence of selenoprotein W (SelW) is necessary to understand the importance of SelW in the antioxidative system. Depletion of SelW by RNA interference was achieved by introducing a synthetic small interfering RNA into the mouse skeletal muscle cell line C2C12 (C3H). Transfectant screening was performed by real-time reverse transcription-PCR, Western blotting, flow cytometry, fluorescence staining, cell viability, and glutathione assays. SelW expression and mRNA levels were downregulated by 62.1 and 72.4%, respectively. In addition, acute cytotoxicity and an apoptosis rate of approximately 36% in SelW-depleted cells demonstrated that RNA interference was successful. As compared with non-SelW-depleted cells, the enzyme activities of glutathione peroxidase, superoxide dismutase, and catalase and total antioxidative capability and glutathione level increased by 47.6, 103.0, 31.0, 205.6, and 30.0%, respectively (P < 0.05). Thus, SelW is important for the antioxidative system of muscle cells. Depletion of SelW, however, could be compensated by other intracellular antioxidative enzymes because oxidative stress was not the causative factor for apoptosis in SelW-depleted cells. Thus, the main function of SelW in muscle cells is not in the antioxidative system.