For multiple low-grade malignant tumors located in the pancreatic head and tail, middle-segment-preserving pancreatectomy (MSPP) is sometimes indicated. However, MSPP has rarely been performed laparoscopically. Here we report the first case of laparoscopic MSPP for multiple pancreatic neuroendocrine tumors diagnosed preoperatively under an endoscopic ultrasound-guided fine-needle aspiration biopsy. A 70-year-old man had multiple small tumors located in the pancreatic head, body and tail. Endoscopic ultrasound-guided fine-needle aspiration biopsy with immunohistochemical staining made a definitive diagnosis of a pancreatic neuroendocrine tumor (G1). To preserve the 5-cm pancreas body, we successfully performed laparoscopic MSPP: subtotal stomach-preserving pancreaticoduodenectomy followed by distal pancreatosplenectomy. Pathological examination revealed negative surgical margin after resection. Postoperative course was uneventful, and at 14 months after the operation, the patient remains tumor-free. The patient has discontinued insulin supplement therapy but does use an oral hypoglycemic agent. Laparoscopy-assisted MSPP, with reconstruction through a 6-cm transverse incision, can be safely performed for selected cases of borderline and malignant lesions.