Ampullary tumors are diagnosed by endoscopic biopsy of the ampulla of Vater. We encountered a rare case of acute pancreatitis following endoscopic biopsy of the ampulla. A 53-year-old man referred to our hospital for detailed examination of a suspected tumor of the ampulla of Vater. We conducted endoscopic biopsy from the ampulla. He developed severe abdominal pain four hours after the procedure. The serum amylase and serum lipase were elevated and abdominal computed tomography (CT) revealed pancreatic enlargement and diffuse stranding of the peri-pancreatic fat, compatible with the findings of acute pancreatitis. We diagnosed the patient as having acute pancreatitis caused by endoscopic biopsy of the ampulla of Vater. Conservative therapy improved his condition, however, a large pancreatic walled-off necrosis (WON) developed. Therefore, we performed endoscopic ultrasonography (EUS)-guided cyst drainage on the 74th day after admission. The WON diminished gradually in size and the symptoms disappeared, and the patient was discharged in good physical condition on the 137th day after admission. In this case, the ampullary biopsy may have caused mucosal edema or intraductal hematoma, resulting in pancreatic duct obstruction. It is important for endoscopists both to be aware of this potential complication following endoscopic biopsy of the ampulla and to inform the patients about possible complications of this procedure.