Hedgehog protein is an essential molecule for gastrointestinal tract development, and disruption of hedgehog signaling pathway is linked to some gastrointestinal tumorigenesis. Here, we performed hedgehog immunostaining in periampullary cancer to evaluate the differences according to the location type of cancer and the differentiation of adenocarcinoma.We retrieved surgical specimens from 43 periampullary cancer patients (15 ampulla of Vater cancer, 12 distal common bile duct cancer, 13 pancreatic head cancer, and 3 combined ampulla of Vater/bile duct cancer). Immunohistochemical stain was performed in both normal and cancerous tissue portions of each case using Sonic hedgehog (H-160) rabbit polyclonal antibody. Immunohistochemical stain results were grouped into three groups according to the percentage of positive cytoplasmic stain in tumor volume (unstained: 50%).All of the normal tissue revealed negative immunohistochemical stain while cancerous tissue revealed positivity in 95.3% (41/43 cases). Strongly stained cases were more frequently seen in ampulla of Vater cancers (13/15) and in combined ampulla of Vater/bile duct cancers (3/3) than in distal common bile duct cancers (4/12) and in pancreatic head cancers (3/13) (p=0.002). In addition, strongly stained cases were more frequently seen in well-differentiated adenocarcinoma than the others (p<0.001).Most of the periampullary cancers show hedgehog protein expression. In addition, hedgehog protein immunostainings shows stronger expression in ampulla of Vater cancers and in well-differentiated adenocarcinoma.