Pancreatic cancer (PC) is characterized by rapid tumor spread, and very few patients with PC survive for more than 5 years. It is imperative to discover additional diagnostic biomarkers or specific therapeutic targets in order to improve the treatment of patients with PC. In search for useful biomarkers, we analyzed ten pairs of non-cancerous and cancer tissues from patients with PC by two-dimensional gel electrophoresis (2-DE). Nineteen protein spots showed differential expression on 2-DE gels between the cancer and non-cancerous tissues. Six upregulated protein spots were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as calreticulin, glutathione synthetase, stathmin, vinculin, α-enolase and glyceraldehyde-3-phosphate dehydrogenase. Western blotting demonstrated that vinculin was predominantly expressed in the pancreatic cancer tissues compared with to non-cancerous tissues. Our findings indicate that vinculin may be a clinically useful biomarker of PC.