The flatworm Fasciola hepatica is responsible for fasciolosis, one of the most common parasitic diseases of livestock worldwide, with increased incidence of human cases. When contaminated plants are ingested, infective larvae are released and transverse the gut wall before migrating to the bile ducts within the liver. Migrating liver flukes erode host tissue while adults feed on blood and they mature and release thousands of eggs. Several developmentally-regulated cathepsin L like proteolytic enzymes (FheCLs) are essential to the migrating and feeding processes. Despite being similar in structure and sequence these enzymes show specialization attacking preferentially different substrates and taking part in the diverse process of invasion, immune evasion and feeding. Our analyses reveal unique differences in activity between the major infective juvenile (FheCL3) and adult (FheCL1) enzymes, and demonstrate that the juvenile enzyme has a particular active site that allows it to degrade collagen, the main component of connective tissues. We demonstrate that a single position on the active site, residue 67, is essential to this collagenolytic activity critical for parasite invasion.