Transgenic engineering is an effective way for plants to obtain strong degradation or detoxification abilities to target pollutants. Acetochlor is an important and widely used herbicide, however, its residue is persistent in soil and is toxic to humans and rotation crops. In this study, the degradation ability and tolerance to acetochlor of transgenic Arabidopsis thaliana synthesizing the oxygenase component, CndA, of the bacterial acetochlor N-dealkylase system, CndABC, were investigated. Two transgenic plants, including a cytoplasm transformant, in which the CndA was located in the cytoplasm, and a chloroplast transformant, in which the CndA was located in the chloroplast, were constructed. The cytoplasm transformant acquired only weak acetochlor degradation activity and displayed little acetochlor tolerance. In contrast, the chloroplast transformant exhibited high degradation efficiency and strong tolerance to acetochlor; it could transform 94.3% of 20?μM acetochlor in water within 48?h and eliminate 80.2% of 5?mg/kg acetochlor in soil within 30 d. The metabolite of acetochlor N-dealkylation catalyzed by CndA, 2-chloro-N-(2-methyl-6-ethylphenyl)acetamide (CMEPA), could be released outside the cells by chloroplast transformant and further degraded by indigenous microorganisms in the soil. This study provides an effective strategy for the phytoremediation of acetochlor residue in water and soil.