In this study, the cellular responses of Stenotrophomonas sp. OK-5 to explosive 2,4,6-trinitrotoluene (TNT) have been extensively analyzed. The stress shock proteins, which might contribute to enhancing cellular resistance to TNT-mediated toxicity, were induced at different concentrations of TNT used as a substrate for cell culture of Stenotrophomonas sp. OK-5 capable of utilizing TNT. Proteomic analysis for 2-DE of soluble protein fractions from the culture of OK-5 exposed to TNT demonstrated approximately 300 spots on the silver-stained gel ranging from pH 3 to pH 10. Among them, 10 spots significantly induced and expressed in response to TNT were selected and analyzed. As the result of internal amino acid sequencing with ESI-Q TOF mass spectrometry, TNT-mediated stress shock proteins such as DnaK, OmpW, and OsmC were identified and characterized. Survival of strain OK-5 was periodically monitored in the presence of different concentrations of TNT along with the production of the stress shock proteins. Cells of strain OK-5 pre-exposed to TNT had in improved survival tolerance. Analysis of total cellular fatty acids in strain OK-5 suggested that several saturated or unsaturated fatty acids might increase or decrease under TNT-mediated stress condition. Scanning electron microscopy of cells treated with 0.8 mM TNT for 12 h revealed irregular rod shapes with wrinkled surfaces.