Type 2 diabetes(T2D) patients are more prone to develop Alzheimer's disease (AD). We have previously shown that Glucagon-like peptide-1 receptor agon- ist exendin-4 (Ex-4) reduces tau hyperphosphorylation in T2D animals through upregulating in- sulin signaling, and peripheral injected Ex-4 increases insulin levels in the T2D brain. This study aims to further clarify whether the elevated insulin in the brain is produced by nerve cells under the action of Ex-4.The neuronal cell line-HT22 was treated with Ex-4 under high glucose or normal cultiva- tion, and the number of insulin-positive cells as well as the expression levels of insulin synthesis-re- lated genes were examined. The db/db mice were treated with a peripheral injection of Ex-4 and/or intracerebroventricular (ICV) injection of siRNA to inhibit the expression of insulin synthesis-relat- ed genes and the behavior tests were carried on. Finally, plasma glucose, cerebrospinal fluid (CSF) glucose, CSF insulin, phosphorylation of tau, phosphorylation of AKT and GSK-3β of db/db mice were detected.We found that Ex-4 promoted the expression of insulin synthesis-related genes and in- duced an obvious increase of insulin-positive HT-22 neuronal cells in a high glucose environment. Peripheral injection of Ex-4 improved the cognitive function of db/db mice and increased brain in- sulin levels which activated brain insulin signaling and subsequently alleviated tau hyperphosphory- lation. However, when siRNA-neurod1 was injected to block insulin synthesis, the cognitive func- tion of db/db mice was not improved under the action of Ex-4 anymore. Moreover, the brain in- sulin levels dropped to an extremely low level, and the phosphorylation level of tau increased signi- ficantly.This study demonstrated that Ex-4 improved cognition function by promoting brain in- sulin synthesis followed by the activation of brain insulin signaling and alleviation of tau hyper- phosphorylation.Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.