Angiostatin, a well-characterized angiostatic agent, is a proteolytic cleavage product of human plasminogen encompassing the first four kringle structures. The fifth kringle domain (K5) of human plasminogen is distinct from angiostatin and has been shown, on its own, to act as a potent endothelial cell inhibitor. We propose that tumor-targeted K5 cDNA expression may act as an effective therapeutic intervention as part of a cancer gene therapy strategy. In this study, we provide evidence that eukaryotically expressed His-tagged human K5 cDNA (hK5His) is exported extracellularly and maintains predicted disulfide bridging conformation in solution. Functionally, hK5His protein produced by retrovirally engineered human U87MG glioma cells suppresses in vitro migration of both human umbilical vein endothelial cells and human macrophages. Subcutaneous implantation of Matrigel-embedded hK5His-producing glioma cells in nonobese diabetic/severe combined immunodeficient mice reveals that hK5His induces a marked reduction in blood vessel formation and significantly suppresses the recruitment of tumor-infiltrating CD45+ Mac3+ Gr1- macrophages. Therapeutically, we show in a nude mouse orthotopic brain cancer model that tumor-targeted K5 expression is capable of effectively suppressing glioma growth and promotes significant long-term survival (>120 days) of test animals. These data suggest that plasminogen K5 acts as a novel two-pronged anticancer agent, mediating its inhibitory effect via its action on host-derived endothelial cells and tumor-associated macrophages, resulting in a potent, clinically relevant antitumor effect.