To defend themselves from attack by pathogens, plants can rely only on their innate defense systems. Defensins are antimicrobial peptides that contribute to plant immunity by displaying a direct cidal activity against various pathogens, some of which are responsible for plant diseases. These determine a significant decrease in the quality and safety of agricultural products, especially among food crops, and cause significant economic losses. There is consequently an increasing interest for antimicrobial compounds such as the defensins, which might be applied in different ways to protect important food or bio-fuel crops. In this review we analyze the techniques that have been reported in the literature for the production of isolated plant defensins of adequate quality and sufficient quantity for potential use in plant protection. For research purposes, defensins have been heterologously expressed in diverse hosts, such as bacteria, yeasts, fungi and plants. Chemical synthesis is instead not commonly used for their production, due to structural characteristics that make it difficult to obtain the correct protein folding. To consider the possibility of producing plant defensins in a large scale, cost-effective methods guaranteeing high quality product are required. Future studies may thus focus on the development of more stable compounds, as well as decreasing production costs by improving preparative syntheses or biotechnological procedures such as using transgenic crops as plant factories.