C1A plant cysteine proteases are synthesized as pre-pro-enzymes that need to be processed to become active by the pro-peptide claves off from its cognate enzyme. These pro-sequences play multifunctional roles including the capacity to specifically inhibit their own as well as other C1A protease activities from diverse origin. In this study, it is analysed the potential role of C1A pro-regions from barley as regulators of cysteine proteases in target phytophagous arthropods (coleopteran and acari). The in vitro inhibitory action of these pro-sequences, purified as recombinant proteins, is demonstrated. Moreover, transgenic Arabidopsis plants expressing different fragments of HvPap-1 barley gene containing the pro-peptide sequence were generated and the acaricide function was confirmed by bioassays conducted with the two-spotted spider mite Tetranychus urticae. Feeding trials resulted in a significant reduction of leaf damage in the transgenic lines expressing the pro-peptide in comparison to non-transformed control and strongly correlated with an increase in mite mortality. Additionally, the analysis of the expression levels of a selection of potential mite targets (proteases and protease inhibitors) revealed a mite strategy to counteract the inhibitory activity produced by the C1A barley pro-prodomain. These findings demonstrate that pro-peptides can control mite pests and could be applied as defence proteins in biotechnological systems.