Plant vacuoles are multifunctional organelles. On the one hand, most vegetative tissues develop lytic vacuoles that have a role in degradation. On the other hand, seed cells have two types of storage vacuoles: protein storage vacuoles (PSVs) in endosperm and embryonic cells and metabolite storage vacuoles in seed coats. Vacuolar proteins and metabolites are synthesized on the endoplasmic reticulum and then transported to the vacuoles via Golgi-dependent and Golgi-independent pathways. Proprotein precursors delivered to the vacuoles are converted into their respective mature forms by vacuolar processing enzyme, which also regulates various kinds of programmed cell death in plants. We summarize two types of vacuolar membrane dynamics that occur during defense responses: vacuolar membrane collapse to attack viral pathogens and fusion of vacuolar and plasma membranes to attack bacterial pathogens. We also describe the chemical defense against herbivores brought about by the presence of PSVs in the idioblast myrosin cell.