Cathepsin proteases are promising vaccine or drug targets for prophylaxis or therapy against Fasciola parasites which express cathepsin L and B proteases during their development. These proteases are believed to be involved in important functions for the parasite, including excystment, migration, feeding and host immune evasion. Several cathepsin L transcripts, including FhCatL5, have been isolated from adult Fasciola, while certain cathepsin L proteases, including FgCatL1G, have only been identified in the juvenile forms of the parasite. In this study, Fasciola hepatica cathepsin FhCatL5 and F. gigantica FgCatL1G were expressed in yeast and their biochemical properties characterised and compared. The pH profiles of activity and stability of the two recombinant cathepsins was shown to differ, differences that are likely to be functionally important and reflect the environments into which the cathepsins are expressed in vivo. Biochemical analysis indicates that FgCatL1G can cleave substrates with proline residues at P2, a characteristic previously described for the adult cathepsin FhCatL2. FgCatL1G and FhCatL5 show differences in their host substrate digestion patterns, with different substrates cleaved at varying efficiencies. Functional analysis of a recombinant FhCatL5 L69W variant indicates that the residue at position 69 is important for the S2 subsite architecture and can influence substrate specificity.