Nestin was first described as an intermediate filament protein expressed in neuroepithelial stem cells. Nestin expression has also been reported in brain tumors, schwannomas, gastrointestinal stromal tumors, and melanomas. In the pancreas, Nestin expression has been detected in exocrine and mesenchymal cells, including stellate cells, pericytes, and endothelial cells. In the present study, we examined Nestin expression in human pancreatic ductal adenocarcinoma and sought to determine its role in this malignancy. Reverse transcription-polymerase chain reaction analysis demonstrated the presence of Nestin mRNA in all 10 tested pancreatic cancer cell lines, and quantitative reverse transcription-polymerase chain reaction revealed that Nestin mRNA levels were highest in PANC-1 cells and lowest in PK-8 cells. Immunofluorescent analysis revealed that Nestin localized in the outer cytoplasm of PANC-1 cells. Nestin immunoreactivity was present in the cancer cells in 20 (33.3%) of 60 cancer cases, and its expression was confirmed by in situ hybridization. Nestin expression was also increased in peripheral nerve fibers adjacent to cancer cells and in peripheral nerve fibers invaded by cancer cells. Clinicopathologically, there was a statistically significant association between Nestin expression in pancreatic cancer cells and nerve invasion (P = .010) and the presence of cancer cells in the tumor resection margins (P = .003). Nestin-positive cases exhibited similar survival after resection by comparison with Nestin-negative cases, irrespective of whether they were given adjuvant therapy. These findings indicate that Nestin expression in pancreatic cancer cells may contribute to nerve and stromal invasion in this malignancy.