Pancreatic ductal adenocarcinoma (PDAC) is a common malignancy with a very low 5-year survival rate. Currently, there are no valid markers for early detection and targets for therapy. Here, we used 2-DE to analyze the protein profiles of eight PDAC specimens and paired adjacent nontumor tissues. MS was used to identify 15 protein spots that were found to be overexpressed in PDAC tissues versus adjacent controls. One of them was identified as phosphoglycerate kinase (PGK) 1, a secretable glycolytic enzyme known to participate in angiogenesis. Immunohistochemical analysis of 63 PDAC specimens revealed moderate to strong expression of PGK1 in >70% of the tumors. Further Western blotting analysis of cells from tumor and adjacent nontumor tissues obtained by laser capture microdissection confirmed the enhanced expression of PGK1 in tumor cells. Furthermore, the serum levels of PGK1 were significantly higher in PDAC patients (n = 21) than in the control group (n = 25) (p < 0.005), as determined by ELISA. These observations indicate that protein profile analysis using a combination of 2-DE and MS provides an effective strategy for identifying biomarkers that may have diagnostic potential for PDAC, and identify PGK1 as a potential biomarker and/or therapeutic target for PDAC.