The mechanism that initiates regeneration of pancreatic β-cells is not clear at present. The vagal nerve is implicated in the regulation of gastrointestinal functions, glucose metabolism and proliferation of pancreatic β-cells under physiological conditions. To elucidate the triggering mechanism of the regeneration of pancreatic β-cells, we examined the involvement of the vagal nerve. To this end, we employed a rat pancreatic duct ligation (DL) model, in which profound β-cell neogenesis and β-cell proliferation were observed within a week. We administered atropine to block the vagal nerve. Administration of atropine inhibited proliferation of β-cells in both islets and islet-like cell clusters (ICC), without affecting ductal cell proliferation in the ligated pancreas. The numbers of PDX-1 and MafB-positive cells in or attaching to the ducts were significantly reduced by atropine. MafB/glucagon and MafB/insulin double-positive cells were also decreased by atropine. Finally, atropine reduced the number of MafA-positive ductal cells, all of which were positive for insulin, by 50% on day 5. These results strongly suggest that the vagal nerve is involved in β-cell proliferation, induction of endocrine progenitors and neogenesis of α- and β-cells.