Plant cystatins have been the object of intense research since the publication of a first paper reporting their existence more than 20 years ago. These ubiquitous inhibitors of Cys proteases play several important roles in plants, from the control of various physiological and cellular processes in planta to the inhibition of exogenous Cys proteases secreted by herbivorous arthropods and pathogens to digest or colonize plant tissues. After an overview of current knowledge about the evolution, structure and inhibitory mechanism of plant cystatins, we review the different roles attributed to these proteins in plants. The potential of recombinant plant cystatins as effective pesticidal proteins in crop protection is also considered, as well as protein engineering approaches adopted over the years to improve their inhibitory potency and specificity towards Cys proteases of biotechnological interest.