The Lon ATP-dependent protease plays an important role in regulating many biological processes in bacteria. In this study, we examined the organic solvent tolerance of a Δlon mutant of Escherichia coli K-12 and found that the mutant showed remarkably higher organic solvent tolerance than the parent strain. Δlon mutants are known to overproduce capsular polysaccharide, resulting in the formation of mucoid colonies. We considered that this increase in capsular polysaccharide production might be involved in the organic solvent tolerance in E. coli. However, a ΔlonΔwcaJ double-gene mutant displaying a nonmucoid phenotype was as tolerant to organic solvents as the Δlon mutant, suggesting that capsular polysaccharide is not involved in organic solvent tolerance. On the other hand, the Lon protease is known to exhibit proteolytic activity against the transcriptional activators MarA and SoxS, which can enhance the expression level of the AcrAB-TolC efflux pump. We found that the Δlon mutant showed a higher expression level of AcrB than the parent strain. In addition, the ΔlonΔacrB double-gene mutant showed a significant decrease in organic solvent tolerance. Thus, it was shown that organic solvent tolerance in the Δlon mutant depends on the AcrAB-TolC pump but not capsular polysaccharide. E. coli strain JA300 acrRIS marR overexpresses the AcrAB-TolC pump and exhibits high-level solvent tolerance. In an attempt to further improve the solvent tolerance of JA300 acrRIS marR, a lon gene disruptant of this strain was constructed. However, the resulting mutant JA300 acrRIS marR Δlon showed lower solvent tolerance than JA300 acrRIS marR.