Dihydropyrazine (DHP), which is produced during the Maillard reaction, generates radicals that not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce oxidative damage to cellular proteins. In the present study, we show that three DHP derivatives, which generated superoxide anions, caused inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). SH-compounds, such as cysteine, dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoethylamine, and N-acetyl-cysteine, suppressed the inhibition of GAPDH by DHP in vitro, although the effect of DHP on GAPDH was not reversed by DTT. In addition, DHP-exposed Escherichia coli showed almost unaffected growth on plates containing a rich medium, but poor growth on plates containing M9 synthetic medium with glucose as the sole carbon source. Furthermore, DHP-exposed E. coli exhibited reduced GAPDH activity. These findings indicate that DHP disturbs the glycolytic pathway by inhibiting GAPDH activity.