Pancreatic cancer (PC) is the sixth leading cause of cancer-associated death worldwide. Despite improvements in treatment methods, the 5-year survival rate of PC patients is still low. Although carbohydrate antigen (CA) 19-9 has been used as the most specific and sensitive biomarker for the diagnosis of PC, approximately 34% of PC patients may have a Lewis antigen-negative phenotype and secrete little, if any, CA19-9. Therefore, it is important to develop effective diagnostic methods for PC. In this study, we performed a global shotgun proteomics analysis using PC cell lines and a normal pancreatic ductal cell line. We identified 142 candidate proteins with differential expression in PC cells. A label-free semiquantitative method based on spectral counting and Gene Ontology analysis led to a total of 25 candidate proteins that could potentially be detected in blood. Validation studies revealed that the expression of fibulin-1 was lower in PC cells than in normal pancreatic ductal cells. Moreover, in vivo fibulin-1 expression was significantly lower in serum from PC patients than in healthy individuals. These findings suggest that lower blood levels of fibulin-1 may be a novel biomarker for the detection of PC.