Reversible thiol oxidation is both a mark of H2O2 toxicity and an initiator of signalling events. H2O2 sensors contain exposed and reactive cysteine residues, which become transiently oxidized as an activation mechanism. In fission yeast, the Pap1 (pombe AP-1) transcription factor has cytosolic localization, and upon H2O2 stress it undergoes post-translational modifications impairing its nuclear export; genetic evidences suggested the formation of a disulfide bond in Pap1 as a triggering activation event. Nuclear Pap1 is then recruited to about 50–80 promoters and induces an adaptation response. We have now dissected the role of all seven cysteine residues in Pap1 using genetic and proteomic techniques, and we show that four of them are required for Pap1 to be activated by H2O2 stress. Thus, mutants lacking each one of these cysteine residues display sensitivity to peroxides. Furthermore, these mutant proteins do not become oxidized by H2O2 and cannot bind to promoters or trigger the Pap1-dependent gene expression program. We also demonstrate by proteomic analysis of reduced and oxidized Pap1 that these four cysteine residues are reversibly oxidized upon H2O2 stress. Our study suggests that not only one but probably two disulfide bonds are required to promote the important conformational changes that promote Pap1 activation and nuclear accumulation.