Plant systems have gained increased attention as an alternative platform for producing heterologous proteins, particularly for industrially relevant proteins. The Cynara cardunculus L. flower extract is traditionally used in cheese production across Mediterranean countries due to its milk-clotting properties. To address the growing demand for plant-based milk-clotting enzymes, we investigated the heterologous production of cyprosin B (CYPB), a key milk-clotting enzyme, in Nicotiana benthamiana. We also examined the role of its plant-specific insert (PSI) domain in enzymatic activity, protein yield and subcellular localisation. Full-length CYPB and a PSI domain-deleted variant (CYPB Delta PSI) were transiently expressed in N. benthamiana leaves using agroinfiltration. Proteins were purified 9 days post-infiltration, yielding similar to 81 mg/kg (CYPB) and similar to 60 mg/kg (CYPB Delta PSI) fresh weight. CYPB Delta PSI showed higher proteolytic activity (similar to 168 IU/mg) than CYPB (similar to 57 IU/mg) and exhibited faster milk-clotting times, suggesting that PSI removal may contribute to enhanced enzymatic efficiency. However, additional factors such as altered glycosylation or localisation may also play a role. Subcellular localisation indicated that CYPB and its PSI domain targeted the vacuole and endocytic vesicles, while CYPB Delta PSI predominantly localised to the endoplasmic reticulum and tonoplast. This suggests that the PSI domain's vital role in vacuolar targeting and membrane permeabilisation ultimately influences protein yield. Our study shows N. benthamiana as a scalable platform for producing recombinant CYPB variants with improved enzymatic activity. It highlights the PSI domain's role in vacuolar sorting without impairing function. These findings contribute to the development of plant-based systems for milk-clotting enzymes for cheese-making.