The role of cholecystokinin (CCK) in the effect of dietary lipid on proximal gastrointestinal function and satiety is controversial. Recent work suggests that fatty acid chain length may be a determining factor. We investigated the mechanism by which long- and short-chain fatty acids activate jejunal afferent nerves in rats. Whole mesenteric afferent nerve discharge was recorded in anaesthetized male Wistar rats during luminal perfusion of saline, sodium oleate, and sodium butyrate (both 10 mM). Both fatty acids evoked characteristic afferent nerve responses, distinct from the mechanical response to saline, that were abolished in rats following chronic subdiaphragmatic vagotomy. The effect of oleate was abolished by the CCK-A receptor antagonist Devazepide (0.5 mg/kg), whereas the effect of butyrate persisted despite pretreatment with either Devazepide or a combination of the calcium channel inhibitors nifedipine (1 mg/kg) and the ω-conotoxins GVIA and SVIB (each 25 μg/kg). In summary, long- and short-chain fatty acids activate intestinal vagal afferents by different mechanisms; oleate acts via a CCK-mediated mechanism and butyrate appears to have a direct effect on afferent terminals.