The aim of this study was to investigate CD99 as a new marker to characterize solid pseudopapillary tumors (SPTs), and to determine a specific panel of markers to identify the disease. We analyzed the clinicopathological characteristics and immunohistochemical features of 37 patients with SPT. All 37 tumors displayed intracytoplasmic dot-like immunoreactivity of CD99 in contrast to membranous staining in all pancreatic endocrine tumors and most of acinar cell carcinomas, along with negative immunostaining in ductal carcinomas. In addition, we observed a loss of expression of E-cadherin in all SPTs as well as in some other pancreatic tumors, and aberrant nuclear expression of β-catenin in most SPTs. Our findings demonstrated for the first time that the pattern of CD99 expression was highly specific for distinguishing SPTs from other pancreatic tumors. CD99 combined with E-cadherin/β-catenin and CD10 can be used as a relatively specific expression profile of SPTs.