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AIM: To clarify the innervation of human gallbladder,with special reference to morphological understanding of gallstone formation after gastrectomy.METHODS: The liver, gallbladder and surrounding structures were immersed in a 10 mg/L solution of alizarin red S in ethanol to stain the peripheral nerves in cadavers (n = 10). Innervation in the areas was completely dissected under a binocular microscope. Similarly,innervation in the same areas of 10 Suncus murinus (S. murinus) was examined employing whole mount immunohistochemistry.RESULTS: Innervation of the gallbladder occurred predominantly through two routes. One was from the anterior hepatic plexus, the innervation occurred along the cystic arteries and duct. Invariably this route passed through the hepatoduodenal ligament. The other route was from the posterior hepatic plexus, the innervation occurred along the cystic duct ventrally. This route also passed through the hepatoduodenal ligament dorsally.Similar results were obtained in S. murinus.CONCLUSION: The route from the anterior hepatic plexus via the cystic artery and/or duct is crucial for preserving gallbladder innervation. Lymph node dissection specifically in the hepatoduodenal ligament may affect the incidence of gallstones after gastrectomy.Furthermore, the route from the posterior hepatic plexus via the common bile duct and the cystic duct to the gallbladder should not be disregarded. Preservation of the plexus may attenuate the incidence of gallstone formation after gastrectomy.