Probing protein function in parasitic flatworms is hampered by the difficulties associated with the development of transgenic approaches. Although RNA interference (RNAi) in schistosomes shows much promise, it has not been reported in other trematodes. Here, we show the successful silencing of the cysteine proteases cathepsin B and L in the infective stage of Fasciola hepatica newly excysted juveniles (NEJs). Silencing resulted in marked reductions in target transcript levels and significant diminution in the encoded proteins in the gut. RNAi of either enzyme in NEJs induced transient, abnormal locomotory phenotypes, and significantly reduced penetration of the rat intestinal wall.