Plants metacaspases mediate programmed cell death in development 1,2, biotic and abiotic stresses 3, damage-induced immune response 4, and resistance to pathogen attack 5. Most metacaspases require Ca2+ for their activation and substrate processing 6–8. However, the Ca2+-dependent activation mechanism remains elusive 9–11. Here we report the crystal structure of Metacaspase 4 from Arabidopsis thaliana (AtMC4) that modulates Ca2+-dependent, damage-induced plant immune defense. The AtMC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of AtMC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca2+. Our analysis provides insight into the Ca2+-dependent activation of AtMC4 and lays the basis for tuning its activity in response to stresses that may help engineer more sustainable crops for production of food and biofuel.