Paragonimus westermani is a trematode parasite, which causes pulmonary and/or extrapulmonary granulomatous disease in humans. Successful invasion of the host tissue is critical for the survival of this tissue-invasive parasite. The enzymatic hydrolysis of host proteins is clearly a prerequisite of this process. In this study, we have investigated the functional roles of the excretory-secretory cysteine proteases of P. westermani newly excysted metacercariae (PwNEM) in tissue invasion. The 27 and 28 kDa enzymes (PwMc27 and PwMc28) purified from PwNEM excretory-secretory products (ESP), preferentially degraded fibrillar proteins, but not globular proteins. PwMc28 significantly facilitated the invasion of PwNEM into mouse peritoneum, whereas a diffusible cysteine protease inhibitor, trans-epoxysuccinyl-L-leuciloamido-(4-guanidino) butane (E-64) inhibited this process dose-dependently. Two distinct isoforms of PwMc28 (PwMc28a and PwMc28b), which exhibited two amino acid differences in their mature domains, were identified by tandem mass spectrometry and sequence analysis. Both enzymes were localized at the tegument on the anterior border and on the oral sucker, which suggests excretion-secretion via exocytosis or via the excretory canal network. The mRNA transcripts of PwMc28a and b were expressed abundantly during the active invasion/migration through the host's tissues, suggesting their relevant function to tissue invasion/migration in the definitive host.