Oxidation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by reactive oxygen species such as HO activate pleiotropic signaling pathways is associated with pathophysiological cell fate decisions. Oxidized GAPDH binds chaperone proteins with translocation of the complex to the nucleus and mitochondria initiating autophagy and cellular apoptosis. In this study, we establish the mechanism by which HO-oxidized GAPDH subunits undergo a subunit conformational rearrangement. HO oxidizes both the catalytic cysteine and a vicinal cysteine (four residues downstream) to their respective sulfenic acids. A 'two-cysteine switch' is activated, whereby the sulfenic acids irreversibly condense to an intrachain thiosulfinic ester resulting in a major metastable subunit conformational rearrangement. All four subunits of the homotetramer are uniformly and independently oxidized by HO, and the oxidized homotetramer is stabilized at low temperatures. Over time, subunits unfold forming disulfide-linked aggregates with the catalytic cysteine oxidized to a sulfinic acid, resulting from thiosulfinic ester hydrolysis via the highly reactive thiosulfonic ester intermediate. Molecular Dynamic Simulations provide additional mechanistic insights linking GAPDH subunit oxidation with generating a putative signaling conformer. The low-temperature stability of the HO-oxidized subunit conformer provides an operable framework to study mechanisms associated with gain-of-function activities of oxidized GAPDH to identify novel targets for the treatment of neurodegenerative diseases.