Complications arising from diabetes mellitus include cognitive deficits, neurophysiological and structural changes in the brain. The current study investigated the expression of cholinergic, insulin, Vitamin D receptor and GLUT 3 in the brainstem of streptozotocin-induced diabetic rats. Radioreceptor binding assays and gene expression were done in the brainstem of male Wistar rats. Our results showed that B-max of total muscarinic, muscarinic M3 receptors was increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. A significant increase in gene expression of muscarinic M3, alpha 7 nicotinic acetylcholine, insulin, Vitamin D-3 receptors, acetylcholine esterase, choline acetyl transferase and GLUT 3 were observed in the brainstem of diabetic rats. Immunohistochemistry studies of muscarinic M1, M3 and alpha 7 nicotinic acetylcholine receptors confirmed the gene expression at protein level. Vitamin D-3 and insulin treatment reversed diabetes-induced alterations to near control. This study provides an evidence that diabetes can alter the expression of cholinergic, insulin, Vitamin D receptors and GLUT 3 in brainstem. We found that Vitamin D-3 treatment could modulate the Vitamin D receptors and plays a pivotal role in maintaining the glucose transport and expressional level of cholinergic receptors in the brainstem of diabetic rats. Thus, our results suggest a therapeutic role of Vitamin D-3 in managing neurological disorders associated with diabetes.