Secreted cysteine proteases are relevant actors in parasite biology, taking part in critical host colonization roles such as traversing tissue barriers, immune evasion and nutrient digestion. In the trematode Fasciola hepatica, the initial step to successful infection of the mammalian host is the excystment of metacercariae and the invasion through the intestinal wall by the newly excysted juveniles (NEJ). While the cathepsin L-like cysteine proteinases secreted by the adult fluke have been extensively characterized, the cataloguing and description of the cathepsins B and L reported in the invasive stages is only sketchy. To identify the cathepsins expressed during excystment and early invasion we constructed cDNA libraries encoding NEJ cathepsins B and L. We found two cathepsin L-like cysteine proteinases (CL3, CL4) and three cathepsins B (CB1, CB2, CB3) which are predominantly expressed in NEJ. Phylogenetic analysis showed that NEJ-expressed cathepsins L constitute a well-defined clade separate from the adult enzymes. Excystment induction resulted in a significant increment in activity towards cathepsin-specific fluorogenic substrates in metacercariae homogenates, consistent with the detection of precursor and mature forms of cathepsins B and L before and after induction. In NEJ culture supernatants, protein and relative activity profiles show subtle changes during the first 48 h, with prevalence of cathepsin L-like activity, although cathepsins CB3 and CL3 were detected by mass spectrometry. Noticeably, the hydrolysis of a substrate with proline in the P2 position was predominant, a property only shared with adult CL2 and vertebrate cathepsin K among the C1A subfamily of cysteine proteases. Collectively these mRNA, protein and enzymatic data demonstrate the existence of a NEJ-specific repertoire of cathepsins expressed early in invasion, distinct to those used by other trematodes, potentially relevant for specific vaccine and chemotherapy design. The diversity of proteases employed by trematodes in the invasion process is discussed.