We provide evidence of a pericellular network of proteases that are elevated and co-expressed in prostate cancer. The network involves the membrane bound serine proteases hepsin and TMPRSS2, the secreted kallikrein-related peptidases KLK4 and KLK14, and the secreted matrix metalloproteinases MMP-3 and MMP-9. Western blot analysis of cell lysates, conditioned cell culture media, immunoprecipitates and cell surface proteins, demonstrates a network of interactions centred largely at the plasma membrane, with the Arg/Lys specific proteases hepsin and TMPRSS2 key regulators of the network. Our data demonstrate that like TMPRSS2, hepsin is able to autoactivate. Active hepsin degrades KLK4, generating a cell associated degradation product with corresponding reduction in levels of cell-free KLK4. In contrast hepsin activates KLK14. TMPRSS2 appears to cleave amino terminal to the KLK4 activation site such that it is available for further processing to generate the active KLK4 protease. In contrast with hepsin, TMPRSS2 degrades KLK14. In addition to these direct mechanisms of regulation, hepsin and TMPRSS2 indirectly modulate KLK4 activity by cleaving the KLK4-activating protease MMP-3. Hepsin and TMPRSS2 also activate MMP-9, which similar to MMP-3, associates with the cell surface. Interestingly our data also show that proteolysis occurs between the membrane spanning and catalytic domains of hepsin and TMPRSS2. Hepsin cleavage occurs via an autoproteolytic mechanism, whereas TMPRSS2 cleavage is mediated by KLK14. Hepsin and TMPRSS2 are not shed from the cell surface but proteolysis likely disrupts domains that regulate the proteolytic activity of these proteases. Immunocytochemical analyses demonstrate that hepsin and TMPRSS2 colocalize on the cell surface with the secreted serine proteases KLK4 and KLK14, only in membrane protrusions, suggesting that reciprocal proteolytic interactions occur in defined cellular structures that are important during cancer dissemination for cell migration, invasion and survival. Also of note, immunohistochemical analysis of serial sections of prostate tumor demonstrated significant overlapping expression of the six proteases in vivo. Collectively these data suggest the possibility that the novel proteolytic network identified by us, will be most important during active dissemination of prostate cancers, and that its disruption could inhibit metastasis.