The purpose of this study was to investigate the distinct roles of advanced glycation end products (AGEs) on insulin-mediated glucose disposal in 3T3-L1 adipocytes and C2C12 skeletal muscle cells. AGE-modified proteins, namely, GO-AGEs, were prepared by incubating bovine serum albumin (BSA) with glyoxal (GO) for 7 days. Glucose utilization rates and the expression of insulin signaling-associated proteins, including Akt, insulin receptor substrate-1, and glucose transporter 4, were determined. GO-AGEs caused insulin resistance (IR) by suppressing insulin-stimulated glucose uptake both in 3T3-L1 adipocytes and C2C12 muscle cells. Interestingly, an unexpected finding was that insulin-stimulated glucose transport in adipocytes was affected by GO-AGEs in a biphasic manner, with an initial steep increase (168%) during the first 8 h of incubation followed by a significantly impaired uptake after extended culture times (24-48 h, p < 0.05). Treatment with GO-AGEs for 24 h markedly accelerated lipid droplet formation compared to the BSA control; however, it was blocked by incubation with an anti-RAGE antibody. Our study suggests that GO-AGEs induce an early dramatic elevation of glucose transport in adipocytes that may be related to the activation of insulin signaling; however, subsequent IR may result from increased oxidative stress and proinflammatory TNF-α production.