Redox signaling is a fundamental mechanism that controls all major biological processes partly via protein cysteine oxidations, including S-glutathionylation. Despite over 2,000 cysteines identified to form S-glutathionylation in databases, the identification of redox cysteines functionally linked to a biological process of interest remains challenging. Here, we demonstrate a strategy combining glutathionylation proteomic database, bioinformatics, and biological screening, which resulted in the identification of S-glutathionylated proteins, including PP2Cα, as redox players of cell migration. We showed that PP2Cα, a prototypical magnesium-dependent serine/threonine phosphatase, is susceptible to S-glutathionylation selectively at non-conserved C314. PP2Cα glutathionylation causes increased migration and invasion of breast cancer cell lines in oxidative stress or upon hydrogen peroxide production. Mechanistically, PP2Cα glutathionylation modulates its protein-protein interactions, activating c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways to elevate migration and invasion. In addition, PP2Cα glutathionylation occurs in response to epidermal-growth factor, supporting a serine/threonine phosphatase PP2Cα as a new redox player in growth factor signal transduction.