Currently, metabolic disorders are one of the major health problems worldwide, which have been shown to be related to perinatal nutritional insults, and the autonomic nervous system and endocrine pancreas are pivotal targets of the malprogramming of metabolic function. We aimed to assess glucose–insulin homeostasis and the involvement of cholinergic responsiveness (vagus nerve activity and insulinotropic muscarinic response) in pancreatic islet capacity to secrete insulin in weaned rat offspring whose mothers were undernourished in the first 2 weeks of the suckling phase. At delivery, dams were fed a low-protein (4% protein, LP group) or a normal-protein diet (20.5% protein, NP group) during the first 2 weeks of the suckling period. Litter size was adjusted to six pups per mother, and rats were weaned at 21 days old. Weaned LP rats presented a lean phenotype (P < 0.01); hypoglycaemia, hypoinsulinaemia and hypoleptinaemia (P < 0.05); and normal corticosteronaemia (P > 0.05). In addition, milk insulin levels in mothers of the LP rats were twofold higher than those of mothers of the NP rats (P < 0.001). Regarding glucose–insulin homeostasis, weaned LP rats were glucose-intolerant (P < 0.01) and displayed impaired pancreatic islet insulinotropic function (P < 0.05). The M3 subtype of the muscarinic acetylcholine receptor (M3mAChR) from weaned LP rats was less responsive, and the superior vagus nerve electrical activity was reduced by 30% (P < 0.01). A low-protein diet in the suckling period malprogrammes the vagus nerve to low tonus and impairs muscarinic response in the pancreatic β-cells of weaned rats, which are imprinted to secrete inadequate insulin amounts from an early age.