Selenoprotein W (SelW) was thought to play an antioxidant role in mammals. Because chicken SelW has no cysteine (Cys) at the residue 37 (Cys37) that is required for the presumed antioxidant function in mammals, this study was conducted to determine whether chicken SelW possessed the same function. Small interfering RNAs (siRNAs) technology was applied to suppress the SelW expression in chicken embryonic myoblasts. Thereafter, these myoblasts were treated with different concentrations of H2O2 and assayed for cell viability, apoptosis rate, reactive oxygen species (ROS) status, and expression levels of apoptosis-related genes and proteins (Bax, Bcl-2, and caspase-3). Silencing of the myoblast SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H2O2. While the knockout down of SelW up-regulated Bax and caspase-3 and down-regulated Bcl-2, the induced oxidative injuries were alleviated by treatment with a ROS scavenger, N-acetyl-l-cysteine (NAC). Chicken SelW protected embryonic myoblasts against cell apoptosis mediated by endogenous and exogenous H2O2. Chicken SelW possesses antioxidant function similar to the mammalian homologues despite the lack of Cys37 in the peptide. Display omitted ► The siRNA suppressed the SelW expression by>77% in chicken myoblasts ► SelW gene silencing enhanced ROS formation in chicken myoblasts ► SelW gene silencing activated cell apoptosis and related signaling ► SelW gene silencing sensitized cells to the H2O2-mediated apoptosis ► Oxidative damages induced by the SelW knockdown was alleviated by NAC