Pancreatic cancer (PC) is one of the most deadly malignant tumors. The aim of this study was to identify potential biomarkers for PC. Using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, the proteomic profiles of pancreatic cancerous and non-cancerous tissues from ten patients with PC were compared. One of the numerous spots that showed stronger intensity in cancerous compared to non-cancerous tissues was identified as non-muscle cofilin (cofilin-1). This up-regulation was validated by Western blot analysis. It is noteworthy that Western blot analysis showed significantly lower expression of muscle cofilin (cofilin-2) in pancreatic cancerous tissues compared to non-cancerous tissues. This is the first time that cofilin isoforms (cofilin-1/2) have been identified to be differentially expressed in pancreatic cancerous tissues. Therefore, cofilin isoforms may serve as candidates for clinically useful biomarkers or therapeutic targets for PC.