The epithelial extracellular serine protease activation cascade involves matriptase (PRSS14) and prostasin (PRSS8), capable of modulating epidermal growth factor receptor (EGFR) signaling. Matriptase activates prostasin by cleaving in the amino-terminal pro-peptide region of prostasin, presumably at the Arg residue of position 44 (R44) of the full-length human prostasin. Using an Arg-to-Ala mutant (R44A) human prostasin, we showed in this report that the cleavage of prostasin by matriptase is at Arg44. This prostasin proteolytic activation site is also cleaved by hepsin (TMPRSS1) to produce active prostasin capable of forming a covalent complex with protease nexin 1 (PN-1). An amino-terminal truncation of EGFR in the extracellular domain (ECD) was observed when the receptor was co-expressed with hepsin. Hepsin and matriptase appear to cleave the EGFR ECD at different sites, while the hepsin cleavage is not affected by active prostasin, which enhances the matriptase cleavage of EGFR. Using hepsin as the prostasin-activating protease in cells co-transfected with EGFR, we showed that active prostasin does not cleave the EGFR ECD directly in the cellular context. Purified active prostasin also does not cleave purified EGFR. Hepsin cleavage of EGFR is not dependent on receptor tyrosine phosphorylation, while the hepsin-cleaved EGFR is phosphorylated at Tyr1068 and no longer responsive to EGF stimulation. The cleavage of EGFR by hepsin does not result in increased phosphorylation of the downstream extracellular signal-regulated kinases (Erk1/2), an event inducible by the matriptase-prostasin cleavage of EGFR. The role of hepsin serine protease should be considered in future studies of epithelial biology concerning matriptase, prostasin, and EGFR.