There is a lack of tools that selectively target vagal afferent neurons (VAN) innervating the gut. We use saporin (SAP), a potent neurotoxin, conjugated to the GI hormone cholecystokinin (CCK-SAP) injected into the nodose ganglia (NG) of male Wistar rats to specifically ablate GI-VAN. We report that CCK-SAP ablates a sub-population of VAN in culture. In vivo, CCK-SAP injection into the NG reduces VAN innervating the mucosal and muscular layers of the stomach and small intestine, but not the colon, while leaving vagal efferent neurons intact. CCK-SAP abolishes feeding induced c-Fos in the NTS, as well as satiation by CCK or glucagon like peptide -1 (GLP-1). CCK-SAP in the NG of mice also abolishes CCK-induced satiation. Therefore we provide multiple lines of evidence that injection of CCK-SAP in NG is a novel selective vagal deafferentation technique of the upper gastrointestinal tract that works in multiple vertebrate models. This method provides improved tissue specificity and superior separation of afferent and efferent signaling compared to vagotomy, capsaicin, and subdiaphragmatic deafferentation.