It is becoming increasingly apparent that insulin signaling regulates neural circuits in the brain, playing roles in the control of appetite, cognition and memory. Interestingly, in humans, intranasal delivery of insulin to specifically target the brain, has an effect on feeding behaviour, glucose regulation and cognition. However, the action of intranasally administered insulin in the brain has not yet been fully explained, especially in respect to food intake. Here, to determine the effect of intranasal insulin in vivo, the mice were placed in metabolic cages after intranasal injection of standard human insulin, the results show that intranasal insulin remarkably reduced food consumption within 2-5 hours after treatment. Then intranasal fluorescence insulin was delivered to track the distribution of insulin receptors in mouse brain to highlight the areas of the brain that intranasal insulin can reach. Results suggest that insulin receptors are located in many areas of the murine brain, including olfactory bulb, hippocampus, brainstem and a novel area nucleus of the horizontal limb of the diagonal band (HDB). Furthermore, knockdown of the insulin receptor in the HDB leads to a significant increase in food intake in male rats but not female rats. Besides, impaired insulin signaling in the HDB also results in hyperactive in female rats but not male rats. Retrograde tracing data show that the HDB receives afferents from the ventral tegmental area (VTA) and mitral cell layer of olfactory bulb, which might be involved in regulating in metabolism. Overall, our data indicates that intranasal delivery of insulin is a good way to target areas of the brain important for controlling energy balance. Due to the metabolic effect that intranasal insulin has both in rodents and humans, further studies are warranted in order to understand which insulin sensitive brain regions and neurons are involved in this process.