D-ribose is transported by high-affinity ribose transporter, encoded by a rbs operon, rbsDACBKR. Although the components of rbs operon are well studied, rbsD is not. When rbsD was overexpressed in the presence of rbsACBK, cell death was observed in D-ribose minimal media. Overexpressed RbsD enhanced the uptake rate of D-ribose. A high level accumulation of methylglyoxal (MG), a toxic compound, was found in the lethal condition. Using NMR spectroscopy and chemical method, we detected the increase of MG in media after treatment of D-ribose. A mutant lacking methylglyoxal synthase (MGS), which is the enzyme of MG formation, was constructed. The MGS mutant grew normally in D-ribose media and failed to produce MG. These results are proposed that one of major factor that affect cell viability is MG production in the cell and RbsD is highly associated with the accumulation of MG. To observe the effect of E. coli MGS in animal cells, we made an expression vector for mgsA gene, which was transfected in HEK-293T cell. The expression of MGS did not affect the growth or survival of HEK-293T cell.