Fasciolosis caused by the liver fluke Fasciola hepatica is a worldwide spread parasitic disease of domestic ruminants responsible for considerable economic losses in the cattle industry. In addition, fasciolosis is now recognized as an emerging human disease. This work is focused on FhCY2, a member of cystatin superfamily encoded by F. hepatica. FhCY2 is expressed from metacercarial to adult stages in several tissues and also found in F. hepatica excretory-secretory products (ESP). Sequence alignment analysis and homology modelling revealed that FhCY2 belongs to the type 1 cystatin (stefin) family, but surprisingly it has also typical features of mammalian members of the type 2 cystatin family, including two disulfide bridges and a signal sequence. Recombinant FhCY2 showed a broad inhibitory specificity towards various mammalian cysteine cathepsins and ESP proteases, including cathepsin L1 (FhCL1), the most abundant ESP protease. FhCL1 was produced by recombinant expression as a stable active-site mutant and used for the preparation of FhCL1-FhCY2 complex for crystallographic analysis. We propose that FhCY2 acts as a physiological regulator of endogenous parasite proteases as well as modulator of the host proteolytic system, and represents a potential vaccination target.