Pancreatic cancer is characterized by early metastatic spread, but the process of tumor cell dissemination is largely unknown. Here, we show that the soluble protein pancreatic adenocarcinoma upregulated factor (PAUF) plays an important role in the metastasis and progression of the disease. Variations in the level of PAUF, either by overexpression or knockdown, resulted in altered migration, invasion, proliferation and adhesion capacity of pancreatic cancer cells. Moreover, depletion of PAUF in metastatic cells dramatically abrogated the spread of the cells to distant organs in an orthotopic xenograft mouse model. We developed human anti-PAUF monoclonal antibody (8F3), which showed an inhibitory effect on metastasis of pancreatic cancer cells in both in vitro and in vivo. PAUF elicited the activation of the ERK, JNK and AKT intracellular signaling cascades and consequently their downstream transcription factors in an autocrine manner. Genome-wide expression analysis revealed that CXCR4 expression was induced by PAUF overexpression but was repressed by PAUF knockdown. The PAUF-mediated increase in cancer cell motility was attenuated by the CXCR4 inhibitor AMD3100 or by anti-CXCR4 antibody. Furthermore, immunohistochemical analysis of pancreatic tumor tissues clearly showed a significant positive correlation between PAUF and CXCR4 expression. Collectively, these findings indicate that PAUF enhances the metastatic potential of pancreatic cancer cells, at least in part, by upregulating CXCR4 expression and suggest that anti-PAUF monoclonal antibody treatment could be used for therapeutic application for pancreatic cancer.