As generalist parasitoid wasps, Leptopilina heterotoma are highly successful on many species of fruit flies of the genus Drosophila. The parasitoids produce specialized multi-strategy extracellular vesicle (EV)-like structures in their venom. Proteomic analysis identified several immunity-associated proteins, including the knottin peptide, LhKNOT, containing the structurally conserved inhibitor cysteine knot (ICK) fold, which is present in proteins from diverse taxa. Our structural and docking analysis of LhKNOT’s 36-residue core knottin fold revealed that in addition to the knottin motif itself, it also possesses a Cation–Polar–Cation (CPC) clip. The CPC clip motif is thought to facilitate antimicrobial activity in heparin-binding proteins. Surprisingly, a majority of ICKs tested also possess the CPC clip motif, including 75 bona fide plant and arthropod knottin proteins that share high sequence and/or structural similarity with LhKNOT. Like LhKNOT and these other 75 knottin proteins, even the Drosophila Drosomycin antifungal peptide, a canonical target gene of the fly’s Toll-NF-kappa B immune pathway, contains this CPC clip motif. Together, our results suggest a possible defensive function for the parasitoid LhKNOT. The prevalence of the CPC clip motif, intrinsic to the cysteine knot within the knottin proteins examined here, suggests that the resultant 3D topology is important for their biochemical functions. The CPC clip is likely a highly conserved structural motif found in many diverse proteins with reported heparin binding capacity, including amyloid proteins. Knottins are targets for therapeutic drug development, and insights into their structure–function relationships will advance novel drug design.