Proteomics combined with cell fractionation was used to identify proteins regulated by high glucose (HG) in human mesangial cells (HMC). Total membrane and cytosolic fraction proteins derived from HMC after 7 days of HG exposure were resolved by a two-dimensional gel electrophoresis approach. DeCyder software was used to analyze the HG-induced protein spot dys-regulation. In the membrane sub-proteome, of the 92 spots that were matched across all gels, HG induced significant down-regulation of only 4 protein spots. The dys-regulated spots from the membrane sub-proteome included Binding Protein (BiP), calreticulin precursor protein, a 63 kDa transmembrane protein from ER/Golgi intermediate and subunit of collagen proline 4-hydroxylase. In the cytosolic sub-proteome, of the 122 spots that were matched across all gels, HG induced down-regulation of 3 protein spots and up-regulation of 2 protein spots significantly. Enolase 1, Annexin VI and ₂-Actin were decreased whereas Heat Shock Protein-70 kD (HSP-70) and Calmodulin (CaM) were increased. Further confocal microscopy and Western immunoblotting of mesangial cells validated the increase in CaM. Immunoblotting of diabetic mouse and rat kidneys exhibited marked increase in CaM at both early and late stages of diabetes, reflecting the potential physiologic relevance of CaM up-regulation. CaM-specific inhibitors blocked glucose transport stimulated by transforming growth factor (TGF)- and insulin in mesangial cells. In conclusion, using a combination of cell-fractionation and protein-expression-profiling we identified a cohort of HG-dysregulated proteins in the HMC and identified a critical and as yet unrecognized role for CaM in glucose transport in mesangial cells.