Current study reports isolation of Cupriavidus strain a3 which can utilize 2-chloro-4- nitrophenol (C4NP) as sole source of carbon and nitrogen, leading to its detoxification. Degradation process was initiated by release of nitrite ion resulting in the formation of 2-chlorohydroquinone as intermediate. The nitrite releasing activity was also evident in the cell free protein extract. Different parameters for 2C4NP biodegradation were optimized. The degradation pattern followed Haldane substrate inhibition model with maximum specific degradation rate (q(max)) of 0.13/h, half saturation constant (Ks) of 0.05 mM, and 2C4NP inhibition constant (Ki) of 0.64 mM. The isolate was successfully applied to remediation of 2C4NP-contaminated soil in microcosm study. 2-Dimensional protein electrophoresis analysis showed that growth of the isolate in the presence of 2C4NP resulted in modification of membrane permeability and induction of signal transduction protein. In our knowledge, this is the first study reporting degradation and detoxification of 2C4NP by Cupriavidus. (C) 2016 Elsevier Ltd. All rights reserved.