Diabetes and cognitive dysfunction are highly prevalent disorders, while the underlying mechanism is still elusive. The effects of Gastrodin on central nervous system have been emphasized recently. In this study, we aim to explore the potential mechanism leading to cognitive dysfunction in diabetes and the therapeutic effect of Gastrodin. Diabetes was induced by a single injection of streptozotocin. RNA sequencing technique was used to identify the potential factors involved. Western blot and immunofluorescence were applied to detect the protein expression. Our results have shown that spatial learning was impaired and hippocampal pyramidal neurons were damaged in diabetic rats, which could be ameliorated by Gastrodin intervention. Transcriptional analysis identified differential expression genes, which were confirmed by qPCR and western blot. Furthermore, p21 activated kinase 2 (PAK2) was selected and its inhibitor could promote the survival of primary hippocampal neurons. It suggested that PAK2 pathway may be involved in cognitive dysfunction in diabetes and a therapeutic target for Gastrodin intervention.