The uPA/uPAR system is known to play a critical role in angiogenesis of glioblastoma. Previously, we have shown that shRNA against uPA and uPAR attenuates angiogenesis by blocking nuclear translocation of angiogenin, inhibition of angiopoietin/Tie2 signaling, and regulating several other pro-angiogenic, angiostatic and anti-angiogenic molecules. Further analysis revealed that GM-CSF, a pleiotropic cytokine, was significantly inhibited in U87MG and 4910 co-cultures with endothelial cells transfected with shRNA against uPA and uPAR. The role of the uPA/uPAR system in this process is not completely understood. Analysis of tumor conditioned medium of U87MG, 4910 and HMECs transfected with shRNA against uPA or uPAR alone or in combination (pU2) revealed inhibition of GM-CSF-enhanced secretion of SVEGFR1 as shown by Western blotting and ELISA. Moreover, phosphorylation of JAK2 and STAT5, the downstream effectors of GM-CSF signaling, was also inhibited in all three cell lines. Phosphorylation at Tyr 166 position of the GM-CSFRβ subunit, the signal activating subunit of the GM-CSF receptor, was inhibited in HMEC, U87MG and 4910 cells. Further analysis revealed that shRNA against uPA and/or uPAR increased secretion of TIMP-1, which is known to enhance SVEGFR1 secretion in endothelial cells. Moreover, addition of purified uPA (with and without GM-CSF) activated JAK2/STAT5 signaling in HMEC. Exogenous addition of SVEGFR1 to pU2 tumor conditioned medium enhanced inhibition of VEGF-induced endothelial capillary tube formation as assessed by an in vitro angiogenesis assay. To determine the significance of these events in vivo, nude mice with pre-established tumors treated with shRNA against uPA and/or uPAR showed decreased levels of GM-CSF and increased levels of SVEGFR1 and TIMP-1 when compared with controls. Enhanced secretion of SVEGFR1 by puPA, puPAR and pU2 in endothelial and GBM cells was mediated indirectly by MMP-7 and augmented by ectodomain shedding of VEGFr1 by tyrosine phosphorylation at the 1213 position. Taken together, these results suggest that the uPA/uPAR system could prove beneficial as an indirect target for inhibition of angiogenesis in glioblastoma.