Involvement of autonomic nervous system (ANS) neurotransmitters on insulin secretion in rats submitted to protein malnutrition during lactation was studied. During the first 2/3 of lactation, mothers received a 4% protein diet (LP). Control group received normal diet (23% protein) (NP). After protein restriction, mothers received normal diets. At 81 days rats were submitted to intravenous glucose tolerance tests (ivGTT). Plasma glucose and insulin concentration (PIC) were measured. Glucose-induced insulin secretion (GIIS) was tested in pancreatic islets. Fasting normoglycemia and hypoinsulinemia were observed in LP rats. Glucose intolerance and low PIC in LP group were detected during ivGTT. Acetylcholine (Ach) or blockage of alpha-adrenoceptors induced high PIC increment in LP rats; atropine or stimulation of alpha-adrenoceptors did not change PIC. Insulin secretion of LP rat islets showed low glucose and carbachol responses. Epinephrine-inhibited GIIS in both islet groups. Hypoinsulinemia observed in lactation-malnourished rats might be caused by alterations in GIIS regulation, including ANS modulation.