Pancreatic juice is a potential source of proteins associated with pancreatic cancer (PC) due to the proximity of ducts to tumor tissue. Therefore, screening of proteins in pancreatic juice from PC patients may identify new PC biomarkers. We analyzed pancreatic juice from patients with pancreatic diseases including PC, chronic pancreatitis (CP) and simple choledocholithiasis (CDS) by 2-DE. Protein spots from PC patients that changed >2-fold compared with both CP and CDS were selected and identified by mass spectrometry (MS). mRNA levels were measured by QRT-PCR in PC cell lines, PC tissues and adjacent pancreatic normal (PN) tissues. Relationships between mRNA levels in PC tissues and their clinical characteristics and promoter methylation were analyzed in PC cell lines and tissues. We found that four proteins were significantly changed in PC compared to CP and simple CDS. Two proteins were up-regulated, serine proteinase-2 (PRSS2) preproprotein and pancreatic lipase-related protein-1 (PLRP1), and two proteins were down-regulated, chymotrypsinogen B (CTRB) precursor and elastase 3B (ELA3B) preproprotein. In all PC cell lines, PRSS 2 mRNA levels were elevated, while PLRP 1 mRNA was detected in 4/5 cell lines. ELA3B mRNA was undetectable in all cell lines, but CTRB mRNA was detected in 2/5 cell lines. In PC tissues compared to PN, levels of PRSS2 mRNA were significantly higher, ELA3B significantly lower, and PLRP1 and CTRB not significantly different. Elevated PRSS2 mRNA levels correlated with high T stage. The ELA3B gene promoter had higher methylation in PC cell lines and tissues compared with PN tissues, and correlated with low ELA3B gene expression. In conclusion, comparative proteomic analysis of pancreatic juice from PC patients is a powerful method to find new PC biomarkers. Hyperexpression of the PRSS2 gene and hypermethylation of ELA3B gene promoter were associated with PC, raising the possibility of their application as new biomarkers in PC diagnosis and screening.