To demonstrate the superoxide anion (O(2)(-)) scavenging activity of thiamine, we comparatively investigated the inhibition of cell growth reduction and repression of the oxidative stress-inducible gene expression (soxS, sodA, zwf and soi-19::lacZ) triggered by paraquat, intracellular O(2)(-) generator, using an Escherichia coli system. When thiamine (>1 μM) was added to the culture, a decrease of growth rate caused by paraquat was significantly recovered. Paraquat treatment (1 μM) to aerobically grown E. coli highly increased the expression of soxS and its regulons sodA and zwf, genes for manganese-containing superoxide dismutase (Mn-SOD) and glucose-6-phosphate dehydrogenase (G6PDH) to cope with the oxidative stress. However, the induction of Mn-SOD and G6PDH was suppressed by the thiamine supplement. The induction of the soi-19::lacZ gene, whose expression was dependent on paraquat, was also repressed by more than 10 μM of the thiamine addition to the culture. To characterize the role of thiamine, which challenges the paraquat toxicity, an in vitro experiment of nitroblue tetrazolium (NBT) reduction was performed. The NBT reduction by O(2)(-) generated in the xanthine/hypoxanthine system was inhibited by the thiamine supplement in a dose-dependent manner. Moreover, it competed with the 2-deoxy-d-ribose in absorbing the hydroxyl radical (OH) generated by γ-irradiation (800 Gy) and thus inhibited the formation of malondialdehyde in vitro. In conclusion, this evidence suggests that thiamine may partly act as an antioxidant to scavenge O(2)(-) (or OH) directly and thus affect the cellular response to oxidative stress induced by reactive oxygen species.