Aims: Protein S -glutathionylation, the formation of a mixed disulfide between glutathione and protein thiols, is an oxidative modification that has emerged as a new signaling paradigm, potentially linking oxidative stress to chronic inflammation associated with heart disease, diabetes, cancer, lung disease, and aging. Using a novel, highly sensitive, and selective proteomic approach to identify S -glutathionylated proteins, we tested the hypothesis that monocytes and macrophages sense changes in their microenvironment and respond to metabolic stress by altering their protein thiol S -glutathionylation status. Results: We identified over 130 S -glutathionylated proteins, which were associated with a variety of cellular functions, including metabolism, transcription and translation, protein folding, free radical scavenging, cell motility, and cell death. Over 90% of S -glutathionylated proteins identified in metabolically stressed THP-1 monocytes were also found in hydrogen peroxide (H ₂ O ₂ )-treated cells, suggesting that H ₂ O ₂ mediates metabolic stress-induced protein S -glutathionylation in monocytes and macrophages. We validated our findings in mouse peritoneal macrophages isolated from both healthy and dyslipidemic atherosclerotic mice and found that 52% of the S -glutathionylated proteins found in THP-1 monocytes were also identified in vivo . Changes in macrophage protein S -glutathionylation induced by dyslipidemia were sexually dimorphic. Innovation: We provide a novel mechanistic link between metabolic (and thiol oxidative) stress, macrophage dysfunction, and chronic inflammatory diseases associated with metabolic disorders. Conclusion: Our data support the concept that changes in the extracellular metabolic microenvironment induce S -glutathionylation of proteins central to macrophage metabolism and a wide array of cellular signaling pathways and functions, which in turn initiate and promote functional and phenotypic changes in macrophages. Antioxid. Redox Signal . 25, 836–851.