The prognosis of patients with pancreatic cancer is very poor because of late diagnosis and the lack of response to various therapies. We tried to identify proteins that might be available for early diagnosis and effective therapies by proteomic profiling of pancreatic cancer tissues. Pancreatic cancerous and paired non-cancerous tissues obtained from surgical resections or autopsies of 10 patients were analyzed by two-dimensional gel electrophoresis. The differential display showed 11 spots whose expression was increased in cancerous tissues compared with the paired non-cancerous tissues. The liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) system identified the spots as alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), triosephosphate isomerase, transgelin, calmodulin, superoxide dismutase(Mn) mitochondrial precursor, glutathione S-transferase P, cyclophilin A, protein disulfide isomerase A3 precursor, and apolipoprotein A-I precursor. Two of the 11 spots were detected as GAPDH. We noticed that 4 of 11 spots were enzymes involved in glycolytic pathway. Increased glycolysis in cancer cells has been regarded as the effect of intratumoral hypoxia and is possibly associated with tumor invasion, metastasis or resistance to therapies. These glycolytic proteins and transgelin, were confirmed by Western blotting and immunohistochemistry.