Alachlor [2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl]acetamide] is a chloroacetanilide herbicide and has been widely used as a selective pre-emergent and post-emergent herbicide to control weeds and grass. Due to its wide usage, direct application on the ground, high solubility in water, and moderate persistence, alachlor and its metabolites have been detected in various environments. Therefore, there is an increasing concern about the environmental fate of alachlor and its metabolites. Microbial biodegradation is a main method of removal of alachlor in the natural environment. In this study, we isolated new alachlor degrading bacterium and proposed a novel alachlor-degrading pathway. The alachlor-degrading bacterial strain, GC-A6, was identified as Acinetobacter sp. using 16S rRNA gene sequence analysis. Acinetobacter sp. GC-A6 utilized alachlor as its sole carbon source and degraded 100 mg L-1 of alachlor within 48 h, which was the highest alachlor degradation efficiency. The degradation pathway of alachlor was studied using GC-MS analysis. Alachlor was initially degraded to 2-chloro-N-(2,6-diethylphenyl) acetamide, which was further degraded to 2,6-diethylaniline and 7-ethylindoline, respectively. 2,6-Diethylaniline was transformed into N-(2,6-diethylphenyl) formamide. N-(2,6-diethylphenyl) formamide was a first-reported intermediate during the degrading pathway of alachlor by single isolate.