Microbial tolerance to organic solvents is critical for efficient production of biofuels. In this study,n-butanol tolerance ofEscherichia coliJM109 was improved by overexpressing of genes encoding stress-responsive small RNA-regulator, RNA chaperone, and molecular chaperone. GenerpoS, coding for sigma S subunit of RNA polymerase, was the most efficient in improvingn-butanol tolerance ofE. coli. The highest OD(600)and the specific growth rate of JM109/pQE80L-rpoS reached 1.692 and 0.144 h(-1)respectively at 1.0% (v/v)n-butanol. Double and triple expression of molecular chaperonesrpoS,secB, andgroSwere conducted and optimized. Recombinant strains JM109/pQE80L-secB-rpoSand JM109/pQE80L-groS-secB-rpoSexhibited the highestn-butanol tolerance, with specific growth rates of 0.164 and 0.165 h(-1), respectively. Membrane integrity, potentials, and cell morphology analyses demonstrated the high viability of JM109/pQE80L-groS-secB-rpoS. This study provides guidance on employing various molecular chaperones for enhancing the tolerance ofE. coliagainstn-butanol.