To identify the location of pancreatic endocrine tumors, arterial stimulation and venous sampling (ASVS) is known to be useful for insulinoma and gastrinoma, but its usefulness for glucagonoma has not been verified to date. Here we report a case of glucagonoma that was diagnosed by ASVS with calcium loading, in which an approximately 6-fold increase of glucagon was observed in the splenic artery territory. MEN1 gene analysis verified the presence of a mutation and the glucagonoma was confirmed after operation. In conclusion, ASVS could be useful for the diagnosis of glucagonoma.