RAPID ALKALINIZATION FACTOR (RALF) peptides are shown to regulate multiple physiological processes in plants, including modulation of immune response in angiosperms. This family of cysteine-rich peptide hormones has considerably expanded during land plant evolution, but the ancestral roles of RALF peptides in stress response remains poorly understood. Here, we used the moss Physcomitrium patens as a model to gain insight into the function of PpRALF peptides in response to abiotic and biotic stress factors in non-vascular plants. The quantitative proteomic analysis revealed cooperative down-regulation of M6 metalloproteases and membrane proteins, including those involved in stress response, in PpRALF1, 2 and 3 knockout (KO) lines.