Gastric cancer (GC) is the fourth most common type of malignant tumor that affects humans worldwide, but few targeted therapies for it have been considered that are based on redox systems. Peroxiredoxin2 (Prx2) functions as a reactive oxygen species (ROS)-mediated signaling regulator that controls H2O2 in mammalian cells, and it is involved in the survival of various malignant tumors. In human GC cells, Prx2 depletion markedly reduced the β-catenin levels and expression of β-catenin target genes and proteins. Cell-based assays demonstrated that Prx2 knockdown significantly ablates the cell viability, invasive activity, and colony-forming ability of both AGS and SNU668 cells. Furthermore, an experiment using conoidinA, a Prx2 inhibitor, revealed that Prx2 inhibition can overcome 5-FU resistance in GC cells. Thus, this study suggests that Prx2 plays a crucial role in regulating Wnt/β-catenin signaling in GC cells.