Aims: To investigate the involvement of osmoprotectant transporters in organic solvent tolerance in Escherichia coli and to construct an E. coli strain with high organic solvent tolerance. Methods and Results: The organic solvent tolerance of Delta betT, Delta proV, Delta proP or Delta putP single-gene knockout mutants of E. coli K-12 strain was examined. Among these mutants, the organic solvent tolerance of the Delta proV mutant remarkably increased compared with that of the parent strain. It has been known that a marR mutation confers tolerance on E. coli to organic solvents. A Delta proV and Delta marR double-gene mutant was more tolerant to organic solvents than the Delta proV or Delta marR single-gene mutant. The n-hexane amount accumulated in E. coli cells was examined after incubation in an n-hexane-aqueous medium two-phase system. The intracellular n-hexane level in the Delta proV and Delta marR double-gene mutant was kept lower than those of the parent strain, Delta proV mutant and Delta marR mutant. Conclusions: The organic solvent tolerance level in E. coli highly increased by dual disruption of proV and marR. Significance and Impact of the Study: This study suggests a new strategy for increasing the organic solvent tolerance level in E. coli to improve the usability of the whole-cell biocatalysts in two-phase systems employing organic solvents.