Mice with a fat-specific insulin receptor knock-out (FIRKO) exhibit a polarization of white adipose tissue into two populations of cells, one small (diameter 50 microm) and one large (diameter >100 microm), accompanied by changes in insulin-stimulated glucose uptake, triglyceride synthesis, and lipolysis. To characterize these subclasses of adipocytes, we have used a proteomics approach in which isolated adipocytes from FIRKO and control (IR lox/lox) mice were separated by size, fractionated into cytosolic and membrane subfractions, and analyzed by sucrose gradient, SDS-PAGE, and mass spectrometry. A total of 27 alterations in protein expression at key steps in lipid and energy metabolism could be defined, which were coordinately regulated by adipocyte cell size, impaired insulin signaling, or both. Nine proteins, including vimentin, EH-domain-containing protein 2, elongation factor 2, glucose-regulated protein 78, transketolase, and succinyl-CoA transferase were primarily affected by presence or absence of insulin signaling, whereas 21 proteins, including myosin non-muscle form A, annexin 2, annexin A6, and Hsp47 were regulated in relation to adipocyte size. Of these 27 alterations in protein expression, 14 changes correlated with altered levels of mRNA, whereas the remaining 13 were the result of changes in protein translation or turnover. These data suggest an intrinsic heterogeneity in adipocytes with differences in protein expression patterns caused by transcriptional and post-transcriptional alterations related to insulin action and cellular lipid accumulation.