Reversible cysteine post-translational modifications serve as a "switch" for protein structure-function dynamics. Herein, we established a strategy for comprehensively mapping the cysteine redoxome by pinpointing oxidized and reduced cysteine residues in the liver of male mice fed a high-fat/high-sucrose diet (HFHSD). We identified 3,151 and 2,361 labeled cysteine residues on 961 and 804 proteins in the normal chow diet and HFHSD groups, respectively. HFHSD shifts the cysteine redox states towards oxidation, especially in the mitochondria. The oxidized and reduced cysteine residues exclusively in the HFHSD group are enriched in cellular redox homeostasis, mitochondrial ATP metabolism, post-translational modifications, and disease-associated pathways. Exclusively reduced cysteine residues in the HFHSD group display a distinct consensus cysteine thiol motif, E[RK]C. The current cysteine redoxome strategy broadens the disease-associated proteome landscape and provides potential therapeutic target cysteine residues critical for regulating protein functions and interactions relevant to pathophysiology.