Phosphorelay signaling of environmental stimuli by two-component systems is prevailing in bacteria and also utilized by fungi and plants. In the fission yeast Schizosaccharomyces pombe, peroxide stress signals are transmitted from the Mak2/3 sensor kinases to the Mpr1 histidine-containing phosphotransfer (HPt) protein and finally to the Mcs4 response regulator, which activates a MAP kinase cascade. Here we show that, unexpectedly, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) physically associates with the Mcs4 response regulator and stress-responsive MAP kinase kinase kinases (MAPKKKs). In response to H2O2 stress, Cys-152 of the Tdh1 GAPDH is transiently oxidized, which enhances the association of Tdh1 with Mcs4. Furthermore, Tdh1 is essential for the interaction between the Mpr1 HPt protein and the Mcs4 response regulator and thus for phosphorelay signaling. These results demonstrate that the glycolytic enzyme GAPDH plays an essential role in the phosphorelay signaling, where its redox-sensitive cysteine residue may provide additional input signals.