ABSTRACTThe signal transduction mechanisms of the oxidative stress response in plants remain largely unexplored. Previously, increased levels of cellular hydrogen peroxide (H2O2) had been shown to drastically affect the plant transcriptome. Genome‐wide transcriptome analyses allowed us to build a comprehensive inventory of H2O2‐induced genes in plants. Here, the primary objective was to determine the subcellular localization of these genes and to assess potential trafficking during oxidative stress. After high‐throughput cloning in Gateway‐derived vectors, the subcellular localization of 49 proteins fused to the green fluorescent protein (GFP) was identified in a transient assay in tobacco (Nicotiana benthamiana) by means of agro‐infiltration and confirmed for a selection of genes in transgenic Arabidopsis thaliana plants. Whereas eight of the GFP‐tagged proteins are exclusively localized in the nucleus, 23 reside both in the nucleus and cytosol, in which several classes of known transcription factors and proteins of unknown function can be recognized. In this study, the mapping of the subcellular localization of H2O2‐induced proteins paves the way for future research to unravel the H2O2 responses in plants. Furthermore, the effect of increased H2O2 levels on the subcellular localization of a subset of proteins was assessed.