To identify new biomarkers that improve the early diagnosis and lead to possible therapeutic targets in pancreatic carcinoma, we performed a proteomic approach to compare serum protein expression patterns of pancreatic carcinoma patients with that of gastric cancer patients, other pancreatic disease patients, and healthy volunteers. By two-dimensional gel electrophoresis (2-DE) analyses and mass spectroscopic identification, 10 protein spots were found significantly changed in pancreatic carcinoma and 5 proteins including cyclin I, Rab GDP dissociation inhibitor beta (GDI2), alpha-1 antitrypsin precursor, Haptoglobin precursor, and Serotransferrin precursor were successfully identified. The increased levels of cyclin I and GDI2 found to be associated with pancreatic carcinoma were further confirmed by Western blot analyses in an independent series of serum samples and/or pancreatic juice samples. Applying immunohistochemistry, we further validated expression of cyclin I and GDI2 in additional pancreatic carcinomas. These results indicate that cyclin I and GDI2 may be potential molecular targets for pancreatic cancer diagnostics and therapeutics.