Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.