(Background and Aim) Bacterial infection underlies the pathogenesis of many human diseases, including acute and chronic inflammation. Recently, we found that subclinical pancreatitis was observed in pancreatic tissue surrounding pancreatic cancer. Herein, we investigated a possible role for bacterial infection in the progression of chronic pancreatitis. (Materials and Methods) Pancreatic juice was obtained from patients with pancreatic cancer (n=20) or duodenal cancer/bile duct cancer (n=16) and subjected to PCR using universal primers for the bacterial 16S ribosomal RNA gene. Bacterial species were identified by PCR using bile samples from four pancreatic cancer patients. Immunohistochemical and serologic analyses for Enterococcus faecalis infection （antibody against Enterococcus faecalis capsular polysaccharide, anti-CPS antibody）were performed on a large cohort of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer and on mice with caerulein-induced chronic pancreatitis. The effect of E. faecalis antigens on cytokine secretion by pancreatic cancer cells was also investigated. (Results) We found that 29 of 36 pancreatic juice samples were positive for bacterial DNA. Enterococcus and Enterobacter species were detected primarily in bile, which is thought to be a pathway for bacterial infection of the pancreas. Enterococcus faecalis was also detected in pancreatic tissue from chronic pancreatitis and pancreatic cancer patients; antibodies to E. faecalis capsular polysaccharide were elevated in serum from chronic pancreatitis patients. Enterococcus-specific antibodies and pancreatic tissue–associated E. faecalis were detected in mice with caerulein-induced chronic pancreatitis. Addition of Enterococcus lipoteichoic acid and heat-killed bacteria induced expression of pro-fibrotic cytokines by pancreatic cancer cells in vitro. (Conclusion) Infection with E. faecalis may be involved in chronic pancreatitis progression, ultimately leading to development of pancreatic cancer.