Abstract: The degradation pathway of the environmental pollutant benzonitrile by the nitrogen-fixing bacterium Ensifer meliloti 1021 was investigated. And the genes encoding the relevant metabolic enzymes were cloned and over-expressed. High performance liquid chromatography (HPLC) analysis revealed that En. meliloti 1021 resting cells degraded benzonitrile to benzamide and benzoic acid. A whole-genome analysis indicated that En. meliloti 1021 lacks nitrilase gene, but contains one nitrile hydratase (NHase) gene and 12 amidase genes. Therefore, the benzonitrile degradation pathway appears to be mediated by the NHase-amidase system. The NHase gene was amplified by PCR and over-expressed in Escherichia coli Rosetta (DE3)cells.The resulting cells degraded 97 mmol·L^-1 benzonitrile in 5 min (90% degradation rate). At 10 min, benzonitrile was undetectable, while 66.9 mmol·L^-1 benzamide was present. A phylogenetic tree including the En.meliloti 1021 amidase genes and three benzamide amidohydrolase genes indicated that four amidase genes clustered with the three benzamide amidohydrolase genes. Gene cloning and over-expression experiments proved that an amidase (Genbank accession number CAC47672.1)exhibits the benzamidase activity responsible for converting benzamide to benzoic acid. Benzamidase comprises 434 amino acids, with a molecular weight of 47 kDa and an isoelectric point of 5.37. The results of this study have a certain theoretical value and may be useful for eliminating benzonitrile from the environment.