Secreted cysteine proteases are major players in host-parasite interactions; in Fasciola hepatica, a distinct group of cathepsins L was found to be predominantly expressed in the juvenile stages, but their enzymatic properties were unknown. Cathepsin L3 (FhCL3) is a main component of the juvenile secretory products and may participate in invasion. To characterize the biochemical properties, the proenzyme was expressed in the methylotrophic yeast Hansenula polymorpha and the mature enzyme was obtained from the culture medium. FhCL3 exhibited optimal activity and stability at neutral pH and a noticeable restricted substrate specificity with 70-fold preference for Tos-Gly-Pro-Arg-AMC over typical cathepsin substrates with hydrophobic or aliphatic residues in the S2 position. Accordingly, FhCL3 efficiently cleaved type I collagen over different pH and temperature conditions, but it did not cleave immunoglobulin. While most cathepsin cysteine proteinases are unable to digest collagen, mammalian cathepsin K, adult F. hepatica FhCL2 and the plant zingipain can also cleave collagen and substrates with Pro in P2 position, but only FhCL3 and zingipain hydrolyze these substrates with the highest efficiency. Molecular modeling and structural comparisons of the collagen cleaving cathepsins indicated that the strong substrate selectivity observed might be due to steric restrictions imposed by bulky aromatic residues at the S2-S3 subsites. The remarkable similarities of the active site clefts highlight the evolutive constrains acting on enzyme function. The presence of a collagen cleaving enzyme in F. hepatica juvenile stages is suggestive of a role in tissue invasion, an essential feature for the establishment of the parasites in their host.