[Objective] Gene arcA encoding arginine deiminase from Pseudomonas putida JUCT1 was integrated into the genome of Escherichia coli JM109(DE3) to enhance its organic solvent tolerance (OST). [Methods] Using genome of P. putida as template, we amplified arcA gene and expressed it in strain E. coli JM109(DE3). Subsequently, arcA gene was integrated into the genome of E. coli JM109(DE3) by red-mediated recombination to obtain a strain with an inheritable OST phenotype. [Results] When growing in 2.0% (V/V) cyclohexane, 0.1% (V/V) toluene, 4.0% (V/V) decalin and 0.1% (V/V) butanol, the OD660 values of recombinant E. coli JM109(DE3)/pET-20b(+)-arcA could reach about 0.8, 0.9, 1.8 and 1.3, respectively. [Conclusion] Our results demonstrate that microbial OST could be enhanced by the expression of arcA gene. Importantly, this work provides experimental data and molecular basis for constructing OST bacteria for industrial applications.