Cuticle-degrading proteases secreted by nematophagous fungi can degrade nematode cuticle during infection. Alkaline proteases from nematode-parasitic fungi show stronger nematicidal activity in vitro than neutral proteases from nematode-trapping fungi. Sequence alignment of these proteases revealed that the active-site residues were much conserved. Disulfide bridges in alkaline proteases not only contribute to the thermal stability of enzyme structure but also increase the flexibility of S1 and S4 pockets located at the substrate-binding site. Molecular electrostatic potential surfaces of these proteases change gradually from negative to positive while arranging in the order from neutral to alkaline proteases, possibly contributing to the distinct extent of substrate (nematode cuticle) attraction by proteases. The differences in flexibility of substrate-binding site and in electrostatic surface potential distribution between neutral and alkaline cuticle-degrading proteases are associated with the changes of their catalytic activities and nematicidal activities with fungal species. Our results indicate that nematode-parasitic and nematode-trapping fungi have evolved for distinct adaptation under selective pressure.