Matrix Metalloproteinase‐9 (MMP‐9) consists of a prodomain, catalytic domain with 3 fibronectin‐like type II modules and C‐terminal hemopexin‐like (PEX) domain. These domains play distinct roles in terms of proteolytic activity, substrate binding and interaction with inhibitors and receptors. To assess the potential of the MMP‐9‐PEX domain to interfere with tumor progression, we stably transfected human glioblastoma cells with an expression vector containing a cDNA sequence of the MMP‐9‐PEX. The selected clones exhibited decreased MMP‐9 activity and reduced invasive capacity. We assessed how secretion of MMP‐9‐PEX by glioblastoma cells affects angiogenic capabilities of human microvascular endothelial cells (HMECs) in vitro. MMP‐9‐PEX conditioned medium treatment caused a reduction in migration of HMECs and inhibited capillary‐like structure formation in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor‐2 protein level. The suppression of HMECs survival by conditioned medium from MMP‐9‐PEX stable transfectants was associated with apoptosis induction characterized by an increase in cells with a sub‐G₀/G₁ content, fragmentation of DNA, caspase‐3, ‐8 and ‐9 activation and poly (ADP‐ribose) polymerase (PARP) cleavage. A significant tumor growth inhibition was observed in intracranial implants of MMP‐9‐PEX stable transfectants in nude mice with attenuation of CD31 and MMP‐9 protein expression. These results demonstrate that MMP‐9‐PEX inhibits angiogenic features of endothelial cells and retards intracranial glioblastoma growth. © 2008 Wiley‐Liss, Inc.