ABSTRACT Cyclotides are a large family of cyclic cystine knot-containing plant peptides that have anthelminthic activities against Haemonchus contortus and Trichostrongylus colubriformis , two important gastrointestinal nematodes of sheep. In this study, we investigated the interaction of the prototypic cyclotide kalata B1 with the external surface of H. contortus larvae and adult worms. We show that cyclotides do not need to be ingested by the worms to exert their toxic effects but that an interaction with the external surface alone is toxic. Evidence for this was the toxicity toward adult worms in the presence of a chemically induced pharyngeal ligature and toxicity of cyclotides toward nonfeeding larval life stages. Uptake of tritiated inulin in ligated adult worms was increased in the presence of cyclotide, suggesting that cyclotides increase the permeability of the external membranes of adult nematodes. Polyethylene glycols of various sizes showed protective effects on the nonfeeding larval life stage, as well as in hemolytic activity assays, suggesting that discrete pores are formed in the membrane surfaces by cyclotides and that these can be blocked by polyethylene glycols of appropriate size. This increased permeability is consistent with recently reported effects of cyclotides on membranes in which kalata B1 was demonstrated to form pores and cause leakage of vesicle/cellular contents. Our data, together with known size constraints on the movement of permeants across nematode cuticle layers, suggest that one action of the cyclotides involves an interaction with the lipid-rich epicuticle layer at the surface of the worm.