Glucose regulates the density and function of 5‐HT₃ receptors on gastric vagal afferent neurones. Since diet‐induced obesity attenuates the responsiveness of gastric vagal afferents to several neurohormones, the aim of the present study was to determine whether high fat diet (HFD) compromises the glucose‐dependent modulation of 5‐HT responses in gastric vagal afferents prior to the development of obesity. Rats were fed control or HFD (14% or 60% kilocalories from fat, respectively) for up to 8 weeks. Neurophysiological recordings assessed the ability of 5‐HT to increase anterior gastric vagal afferent nerve (VAN) activity in vivo before and after acute hyperglycaemia, while electrophysiological recordings from gastric‐projecting nodose neurones assessed the ability of glucose to modulate the 5‐HT response in vitro. Immunocytochemical studies determined alterations in the neuronal distribution of 5‐HT₃ receptors. 5‐HT and cholecystokinin (CCK) induced dose‐dependent increases in VAN activity in all rats; HFD attenuated the response to CCK, but not 5‐HT. The 5‐HT‐induced response was amplified by acute hyperglycaemia in control, but not HFD, rats. Similarly, although 5‐HT induced an inward current in both control and HFD gastric nodose neurones in vitro, the 5‐HT response and receptor distribution was amplified by acute hyperglycaemia only in control rats. These data suggest that, while HFD does not affect the response of gastric‐projecting vagal afferents to 5‐HT, it attenuates the ability of glucose to amplify 5‐HT effects. This suggests that glucose‐dependent vagal afferent signalling is compromised by short periods of exposure to HFD well in advance of obesity or glycaemic dysregulation.