Intestinal detection of nutrients is a crucial step to inform the whole body of the nutritional status. In this paradigm, peripheral information generated by nutrients is transferred to the brain, which in turn controls physiological functions, including glucose metabolism. Here, we investigated the effect of enteric glucose sensors stimulation on hypothalamic nitric oxide (NO) release in lean or in obese/diabetic (db/db) mice. By using specific NO amperometric probes implanted directly in the hypothalamus of mice, we demonstrated that NO release is stimulated in response to enteric glucose sensors activation in lean but not in db/db mice. Alteration of gut to hypothalamic NO signaling in db/db mice is associated with a drastic increase in inflammatory, oxidative/nitric oxide (iNOS, IL-1β), and endoplasmic reticulum stress (CHOP, ATF4) genes expression in the jejunum. Although we could not exclude the importance of the hypothalamic inflammatory state in obese and diabetic mice, our results provide compelling evidence that enteric glucose sensors could be considered as potential targets for metabolic diseases.