Succinonitrile, an environmental pollutant, has been proved to be degraded by Klebsiella oxytoca. However, the growth rate of this bacterium was decreased by succinonitrile. The aim of this study is to determine the effects of succinonitrile on the physiological responses of K. oxytoca were investigated. Using two-dimensional polyacrylamide gel electrophoresis (2-DE) and MALDI-TOF-MS, we identified the 7 differentially expressed proteins in K. oxytoca treated with succinonitrile in comparison with the cells treated with ammonia. The decreased expression of one protein, ribosomal protein L9, was involved in the biosynthesis pathway of the functional ribosome. The increased expression of proteins such as superoxide dismutase (SOD), glutathione-S-transferase (GST), and PsaA, a metal binding protein, might be involved in the detoxification process of succinonitrile, which suggested that the biodegradation of succinonitrile was accompanied by the occurrence of oxidative stress. Thus, oxidative stress plays a major role in succinonitrile toxicity in vivo, and cellular defense against this toxicity involves activation of antioxidant proteins. Although succinonitrile biodegradation by K. oxytoca is limited by the toxicity of the compound, minimizing oxidative stress during this biodegradation and bolstering the capacity of K. oxytoca to deal with this stress will improve their effectiveness in succinonitrile biodegradation. With the help of proteomic analysis, we can understand about the toxic mechanism of succinonitrile and/or gain more information and deeper understanding about the defense mechanism of K. oxytoca. (C) 2008 Elsevier Ltd. All rights reserved.