Pathological alterations in glomerular mesangial cells play a critical role in the development of diabetic nephropathy, the leading cause of end-stage renal disease. Molecular mechanisms mediating such alterations, however, remain to be fully understood. The present study first examined the effect of high glucose on the mRNA expression profile in rat mesangial cells using cDNA microarray. Based on variation-weighted criteria and with a false discovery rate of 4.3%, 459 of 17,664 cDNA elements examined were found to be upregulated and 151 downregulated by exposure to 25 mM d-glucose for 5 days. A large number of differentially expressed genes belonged to several functional categories, indicating high glucose had a profound effect on mesangial cell proliferation, protein synthesis, energy metabolism, and, somewhat unexpectedly, protein sorting and the cytoskeleton. Interestingly, several thiol antioxidative genes (glutathione peroxidase 1, peroxiredoxin 6, and thioredoxin 2) were found by microarray and confirmed by real-time PCR to be upregulated by high glucose. These changes suggested that the oxidative stress known to be induced in mesangial cells by high glucose might be buffered by upregulation of the thiol antioxidative pathway. Upregulation of thiol antioxidative genes also occurred in high-glucose-treated human mesangial cells and in glomeruli isolated from rats after 1 wk of streptozotocin-induced diabetes, but not in human proximal tubule cells. High glucose slightly increased lipid peroxidation and decreased the amount of reduced thiols in rat and human mesangial cells. Disruption of the thiol antioxidative pathway by two different thiol-oxidizing agents resulted in a three- to fivefold increase in high-glucose-induced lipid peroxidation. In summary, the present study provided a global view of the short-term effect of high glucose on mesangial cells at the level of mRNA expression and identified the upregulation of the thiol antioxidative pathway as an adaptational response of mesangial cells to high glucose.