There are few reports describing the diagnostic significance of S100A6 expression in clinical samples obtained from patients with pancreatic disease. In the present study, we measured S100A6 expression in pancreatic tissues and juice to evaluate its involvement in pancreatic carcinogenesis. We did quantitative real-time reverse transcription-PCR to measure mRNA expression in microdissected cells and pancreatic juice samples. Microdissected invasive ductal carcinoma and intraductal papillary mucinous neoplasm (IPMN) cells expressed significantly higher levels of S100A6 than did microdissected pancreatitis-affected epithelial and normal cells (all comparison; P < 0.008). Median levels of S100A6 in invasive ductal carcinoma were higher than those in IPMN, and those in pancreatitis-affected epithelial cells tended to be higher than those in normal cells, although these differences were not statistically significant. In analyses of pancreatic juice, IPMN and pancreatic cancer samples expressed significantly higher levels of S100A6 than did chronic pancreatitis samples (both; P < 0.017), but levels in pancreatic cancer and IPMN samples did not differ form each other. Receiver operating characteristic (ROC) curve analysis revealed that measurement of S100A6 was useful for discriminating cancer (area under the ROC curve, 0.864) or IPMN (area under the ROC curve, 0.749) from chronic pancreatitis. The present data suggest that expression of S100A6 is increased in a stepwise manner during pancreatic carcinogenesis and may be a biomarker for evaluating malignant potential. Measurement of S100A6 in pancreatic juice may be useful to detect early pancreatic cancer or identify individuals with high-risk lesions that may progress to pancreatic cancer.