To capture prey, onychophorans (velvet worms) expel a slime that forms stiff fibers upon shearing and dehydration. The high quantities of phosphorus previously found in the slime of the velvet worm Euperipatoides rowelli were ascribed to protein phosphorylation. We provide clear evidence, instead, that it is primarily present as phosphonate moieties in the slime of representative from both major onychophoran subgroups which diverged ~380 MYA. Advanced NMR and mass spectrometry demonstrate that 2-aminoethyl phosphonate (2-AEP) is associated with high molecular weight slime proteins as phosphonoglycans. Biogenic phosphonates are a substantial component of the organophosphorus cycle in marine environments but were not previously reported in terrestrial invertebrates. The evolutionary conservation of this rare protein modification suggests a potential role in the formation and function of these biological adhesive fibers with implications for bio-inspiration.