Trypsinogen/trypsin is one of the major serine proteases and is produced by pancreatic acinar cells. Tumor-associated trypsinogen (TAT) has been reported to be produced by several cancer cell lines. The biological roles and activation mechanisms of both TAT and pancreatic acinar trypsinogen (PAT) have not been elucidated in the context of cancer extension, in particular at the stage of invasion and metastasis. In this study, we investigate the roles played by PAT and TAT in pancreatic cancer invasion. In addition, we determined their mechanisms of activation and identified a trypsinogen activity-stimulating factor (TASF) produced by pancreatic cancer cells. TAT expression and high TAT activity were associated with high invasive and liver metastatic potential in SW1990 and CAPAN-2 cells. Moreover, a trypsinogen activating effect and activity prolonging effect was observed in a mixture of these supernatants with trypsinogen. These cells revealed significantly enhanced invasiveness upon invasion assay and in the presence of PAT. TAT and PAT were activated by TASF, active u-PA, produced by pancreatic cancer cells. Activated TAT and PAT can degrade not only ECM proteins but they can also activate other latent proteases. This ECM-protease-network may form a vicious cycle, thereby promoting tumor cell invasion.