We examined the hypothesis that activation of the apoptosis cascade occurs relatively early in diabetes mellitus affecting three distinct neuronal populations that are involved in regulating gut function: (i) dorsal root ganglion (DRG), (ii) vagus nodose ganglion and (iii) colon myenteric plexus. A validated streptozotocin‐induced diabetic rat model and age‐matched healthy controls were studied. After 4–8 weeks of diabetes the animals were anaesthetized, fixed in situ and the relevant tissues removed. After 1 month of diabetes some animals were treated with insulin for 2 weeks to restore euglycaemia. Apoptosis was measured using immunohistochemical detection of activated caspase‐3 and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling (TUNEL)‐positive cells in adjacent sections in neurones (PGP 9.5‐positive cells). The level of apoptosis was confirmed using double‐label assessment of caspase‐3 and TUNEL in DRG preparations. Caspase‐3 immunoreactive neurones demonstrated a range in staining intensity. When all grades of staining were included, 6–8% of the DRG, nodose ganglia and myenteric neurones were immunoreactive in the preparations from diabetic rats compared with 0.2–0.5% in controls. Neurones staining positive for both caspase‐3 and TUNEL accounted for 1–2% of the total neuronal population in all three preparations in diabetic rats compared with 0.1–0.2% in controls (P < 0.05). Insulin treatment reversed the percentage of TUNEL‐positive neurones in diabetic rats to control levels. Activation of the apoptosis cascade occurs relatively early in diabetic autonomic neuropathy and may contribute to the pathophysiology of this disorder.