In this study, the enhanced degradation of TNT using cultures of genome-shuffled Stenotrophomonas maltophilia OK-5 mt-3 has been examined and the proteome of shuffled strain was compared to the wild-type OK-5 strain. Genome shuffling of S. maltophilia OK-5 was used to achieve a rapid enhancement of TNT degradation. The initial mutant population was generated by NTG treatment and UV irradiation. The wild-type OK-5 strain was able to degrade 0.2 mM TNT within 6 days, yet barely tolerated 0.5 mM TNT while the shuffled OK-5 mt-3 was capable of completely degrading 0.5 mM TNT within 8 days, and 1.2 mM within 24 days. The proteomic analysis of the shuffled OK-5 mt-3 demonstrated the changes in the expression levels of certain proteins compared to wild-type OK-5. These results provide clues for understanding TNT tolerance and improved TNT degradation by shuffled S. maltophilia OK-5 mt-3 and have possible applications in the processing of industrial waste containing relatively high TNT concentrations.