Glutathione peroxidase 4 (GPx4) is a monomeric selenium-dependent glutathione peroxidase highly expressed in mammalian cells, which can reduce phospholipid hydroperoxides. However, it has been difficult to express recombinant mammalian GPx4 in Escherichia coli because of the differences in the selenocysteine (Sec) incorporation machinery between eukaryotes and prokaryotes. In this study, an E. coli BL21(DE3)cys auxotrophic strain was used to express GPx4 mutants. We found that untargeted substitution of Cys-2, Cys-37, Cys-75, Cys-107, and Cys-148 with Sec led to loss of activity, suggesting that mutation of any of these Cys residues in GPx4 could result in a structural change. Additionally, we found that the catalytic activity of GPx4 mutants increased as the number of noncatalytic Sec residues decreased, indicating that the negative effects could be mitigated by replacing these Cys residues with Ser residues. A GPx4 mutant with all Cys residues converted to Ser exhibited a “Ping–Pong” mechanism and structure similar to that of native GPx4, indicating that it could act as a substitute for GPx4, when heterologously expressing the protein in E. coli. This research provides an important foundation for biosynthesis of selenium-dependent GPx4 mutants in E. coli.