Physical damage to cells leads to the release of immunomodulatory peptides to elicit a wound defense response in the surrounding tissue. In Arabidopsis thaliana, the plant elicitor peptide 1 (Pep1) is processed from its protein precursor, PRECURSOR OF PEP1 (PROPEP1). We demonstrate that upon damage, both at the tissue and single-cell levels, the cysteine protease METACASPASE4 (MC4) is instantly and spatiotemporally activated by binding high levels of Ca2+ and is necessary and sufficient for Pep1 maturation. Cytosol-localized PROPEP1 and MC4 react only after loss of plasma membrane integrity and prolonged extracellular Ca2+ entry. Our results reveal that a robust mechanism consisting of conserved molecular components links the intracellular and Ca2+-dependent activation of a specific cysteine protease with the maturation of damage-induced wound defense signals.