Abstract: Plant peroxisomes have the capacity to generate different reactive oxygen and nitrogen species (ROS and RNS), such as H₂O₂, superoxide radical (O₂· ⁻), nitric oxide and peroxynitrite (ONOO^‐). These organelles have an active nitro-oxidative metabolism which can be exacerbated by adverse stress conditions. Hydrogen sulfide (H₂S) is a new signaling gasotransmitter which can mediate the posttranslational modification (PTM) persulfidation. We used Arabidopsis thaliana transgenic seedlings expressing cyan fluorescent protein (CFP) fused to a canonical peroxisome targeting signal 1 (PTS1) to visualize peroxisomes in living cells, as well as a specific fluorescent probe which showed that peroxisomes contain H₂S. H₂S was also detected in chloroplasts under glyphosate-induced oxidative stress conditions. Peroxisomal enzyme activities, including catalase, photorespiratory H₂O₂-generating glycolate oxidase (GOX) and hydroxypyruvate reductase (HPR), were assayed in vitro with a H₂S donor. In line with the persulfidation of this enzyme, catalase activity declined significantly in the presence of the H₂S donor. To corroborate the inhibitory effect of H₂S on catalase activity, we also assayed pure catalase from bovine liver and pepper fruit-enriched samples, in which catalase activity was inhibited. Taken together, these data provide evidence of the presence of H₂S in plant peroxisomes which appears to regulate catalase activity and, consequently, the peroxisomal H₂O₂ metabolism.