HO-oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalytic cysteine residues (C(SH) undergo rapid S-glutathionylation. Restoration of the enzyme activity is accomplished by thiol/disulfide S2 displacement (directly or enzymatically) forming glutathione disulfide (G(SS)G) and active enzyme, a process that should be facile as C(SH) reside on the subunit surface. As S-glutathionylated GAPDH accumulates following ischemic and/or oxidative stress, in vitro/silico approaches have been employed to address this paradox. C(SH) residues were selectively oxidized and S-glutathionylated. Kinetics of GAPDH dehydrogenase recovery demonstrated that glutathione is an ineffective reactivator of S-glutathionylated GAPDH compared to dithiothreitol. Molecular dynamic simulations (MDS) demonstrated strong binding interactions between local residues and S-glutathione. A second glutathione was accommodated for thiol/disulfide exchange forming a tightly bound glutathione disulfide G(SS)G. The proximal sulfur centers of G(SS)G and C(SH) remained within covalent bonding distance for thiol/disulfide exchange resonance. Both these factors predict inhibition of dissociation of G(SS)G, which was verified by biochemical analysis. MDS also revealed that both S-glutathionylation and bound G(SS)G significantly perturbed subunit secondary structure particularly within the S-loop, region which interacts with other cellular proteins and mediates NAD(P) binding specificity. Our data provides a molecular rationale for how oxidative stress elevates S-glutathionylated GAPDH in neurodegenerative diseases and implicates novel targets for therapeutic intervention.