We previously showed the important role of glutathione (GSH) in the protection mechanism against different stresses, such as acid pH, saline, and oxidative stress, using a GSH-deficient mutant of Bradyrhizobium sp. (peanut microsymbiont). In this work, we studied the role of GSH in the protection mechanism against methylglyoxal (MG) toxicity. MG is a naturally occurring toxic electrophilic compound, and it has been shown that GSH is involved in the detoxification of MG in Escherichia coli. One recognized component of this detoxification process is the formation of a GSH adduct, which in turn transports potassium (K+) out of bacterial cells. Our results showed that growth of wild-type strain Bradyrhizobium sp. SEMIA 6144 was not affected at a MG concentration of 0.5 mM in the yeast extract-mannitol culture medium. However, a reduction of growth, at concentrations of 1.5 and 2.5 mM MG and reaching complete growth inhibition at 3.0 mM MG, was observed. In wild-type strain, intracellular GSH content decreased, and intracellular K+ content was unchanged, whereas GSH-deficient mutant SEMIA 6144-S7Z was unable to grow at 1.5 mM MG. The addition of external GSH to the incubation medium did not restore the growth rate either in wild-type or mutant strains. Our findings showed that GSH has not proven to be protective against the cell-growth inhibiting activity of MG. Therefore, the response of Bradyrhizobium sp. growth to MG is different from that reported in E. coli and other Gram-negative bacteria.