Pancreatic adenocarcinoma upregulated factor (PAUF) was recently reported to be a metastasis factor for pancreatic cancer cells. Here, we demonstrate a novel role for PAUF as a potent endothelial activator, promoting both angiogenesis and vascular permeability. Overexpression of PAUF in a mouse pancreatic cancer model resulted in increased tumor vascularity. Recombinant PAUF (rPAUF) enhanced proliferation, migration and capillary-like tube formation of human endothelial cells (ECs), consistently with increased neovascularization in vivo. rPAUF also increased endothelial permeability through the disruption of vascular endothelial-cadherin-facilitated cell-cell junctions in vitro and induced vascular leakage in mouse skin. These effects were attenuated upon treatment with an antibody against PAUF. Moreover, PAUF evoked a time- and dose-dependent activation of extracellular signal-regulated kinase (ERK)1/2, AKT and endothelial NO synthase (eNOS) in ECs, which are closely linked to rPAUF-induced angiogenesis. Finally, rPAUF upregulated the expression of C-X-C chemokine receptor 4 (CXCR4) in ECs and potentiated the in vitro and in vivo EC angiogenic responses to stromal cell-derived factor-1 (SDF-1), a ligand for CXCR4. Taken together, these data demonstrate that PAUF has a novel function in promoting angiogenesis and vascular permeability. Our findings suggest new possibilities for PAUF's role in the pathogenesis of angiogenesis-dependent diseases.