Given the high fatality rate of pancreatic cancer, an effective treatment for this devastating disease is urgently needed. We have shown that mesothelin expression was higher in human pancreatic cancer cells than in human pancreatic duct epithelial cells, and mesothelin mRNA was substantially overexpressed in 18 of 21 (86%) clinical pancreatic adenocarcinoma specimens when compared with the surrounding normal tissues. However, the biological functions of mesothelin in tumor progression are not clearly understood. Here we studied the effects of mesothelin overexpression in pancreatic cancer cell proliferation and migration in vitro and pancreatic cancer progression in vivo. We found that forced expression of mesothelin significantly increased tumor cell proliferation and migration by 90% and 300%, respectively, and increased tumor volume by 4-fold in the nude mice xenograft model when compared with the vector control cell line. Silencing of mesothelin inhibited cell proliferation and migration in pancreatic cancer cells and ablated tumor progression in vivo. Vaccination with chimeric virus-like particles that contain human mesothelin substantially inhibited tumor progression in C57BL/6J mice. The increases in mesothelin-specific antibodies and CTL activity and the decrease in regulatory T cells correlated with reduced tumor progression and prolonged survival. This study revealed novel functions of mesothelin and suggested a new therapeutic vaccine strategy whereby mesothelin is targeted to control pancreatic cancer progression. [Mol Cancer Ther 2008;7(2):286–96]