EphB4 is a member of the largest family of receptor tyrosine kinases and is commonly over-expressed in most epithelial cancers, including 66% of prostate cancers where it has been shown to promote tumour angiogenesis, increase cancer cell survival and facilitate cell invasion and migration. Recent studies have suggested that this is via a ligand-independent process, but how the EphB4 protein is regulated at the surface of the prostate cancer cell is unclear. We have identified prostate cancer-associated EphB4 cleavage events using an EphB4-over-expression model of the prostate cancer cell line 22Rv1 and have identified the kallikrein-related peptidase 4 (KLK4) as a possible mediator of this cleavage.  KLK4 is a serine protease that is commonly elevated in prostate cancers, with strong expression seen in tumours that have metastasised to the bone.  The ability of KLK4 to cleave EphB4 was confirmed using recombinant proteins.  Recombinant KLK4 was also used to demonstrate cleavage of EphB4 present on the surface of prostate cancer cells.  The primary cleavage site was determined by N-terminal sequencing to be after R507, in the juxtamembrane extracellular domain, consistent with the identified fragments of 70 and 50 kDa. A second C-terminal fragment of 47 kDa was also generated, possibly as a consequence of ectodomain shedding, with preliminary evidence suggesting this is via the action of the intracellular protease, γ-secretase. This study has not only revealed a new substrate for the KLK4 protease, whose strong link to prostate cancer has been long known, but whose mechanistic contribution still remains poorly understood, but has also identified a possible mechanism for the regulation of EphB4 signaling in prostate cancer and therefore the regulation of the ligand-independent tumour progressive actions of EphB4.