Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal (4-ONE) implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomic technologies were utilized to identify aldehyde-modified proteins in adipose tissue. Adipose proteins from lean insulin sensitive and obese insulin resistant C57Bl/6J mice were reacted with biotin hydrazide and detected using HRP-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a ~2-3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased ~3-4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-sepharose affinity chromatography, proteolytically digested and subjected to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Interestingly, enzymes involved in cellular stress response, lipotoxicity and insulin signaling such as glutathione-S-transferase M1, peroxiredoxin 1, glutathione peroxidase, eukaryotic elongation factor 1a1 (eEF1a1), and filamin A were identified. The adipocyte fatty acid-binding protein (A-FABP), a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of A-FABP with 4-HNE was mapped to Cys117, occurred equivalently using either the (R) or (S) enantiomer of 4-HNE and reduced the protein’s affinity for fatty acids ~10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance.