Intestinal glucagon-like peptide-1 (GLP-1) and pancreatic insulin, released postprandially, commonly regulate glucose metabolism. Recent clinical experience indicates that the GLP-1R agonist and insulin in combination, compared to insulin alone, results in better glycemic and weight controls in type 2 diabetic patients. These observations suggest possible interactive effect of these hormones. These hormones, in addition to peripherally controlling glycemia, exert central regulation of food intake and glucose metabolism, the effect at least partly mediated by signaling to the brain via the vagal afferents. However, whether the vagal afferents are involved in the interactive effects of GLP-1 and insulin remains unknown. The present study explored possible cooperative effect of GLP-1 and insulin on vagal afferent neurons isolated from nodose ganglion (NG) of mice, while monitoring the neuronal activity by measuring cytosolic Ca2+ concentration ([Ca2+]i) with fura-2. GLP-1 at 10-8M increased [Ca2+]i in 8-11% of single NG neurons. GLP-1-induced [Ca2+]i increases were inhibited by GLP-1 receptor antagonist exendin (9-39). Majority (92%) of GLP-1-responseive NG neurons also responded to 10-7M insulin with [Ca2+]i increases. Both GLP-1 and insulin at lower concentration of 10-9M induced [Ca2+]i increases with smaller amplitude in lesser NG neuron population (4-7%). These hormones at 10-9M in combination recruited the unresponsive neurons to [Ca2+]i increases, and induced [Ca2+]i increases with greater amplitude in the responsive neurons. The results demonstrate that GLP-1 and insulin synergistically and additively activate vagal afferent neurons. This interaction may be linked to the postprandial functions mediated commonly by GLP-1 and insulin and in the beneficial outcome of the therapy with GLP-1 receptor agonist and insulin in combination.