Type 2 diabetes is a risk factor for late-onset Alzheimer's disease. However, the underlying mechanisms remain unknown. To investigate whether insulin is associated with the assembly of amyloid beta-protein from the cell surface, we treated nerve growth factor (NGF)-treated rat pheochromocytoma 12 (PC12) cells with insulin, which is related to the development of diabetes. Insulin treatment induced a decrease in GM1 ganglioside (GM1) levels in detergent-resistant membrane microdomains of NGF-treated PC12 cells. The insulin-induced effects on GM1 levels were regulated by a phosphatidylinositol 3-kinase inhibitor, but not by an extracellular signal-regulated kinase inhibitor. Pre-treatment with a protein synthesis inhibitor did not inhibit the decrease in GM1 levels induced by insulin. In addition, insulin failed to induce formation of fibrils from soluble amyloid beta-protein or to accelerate GM1-induced fibril formation. Furthermore, assembly of amyloid beta-protein in cultures of NGF-treated PC12 cells was significantly decreased by insulin. These results suggest that insulin inhibits amyloid beta-protein assembly by decreasing GM1 expression in detergent-resistant membrane microdomains of neuronal membranes.