A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analysis indicated the major pathway of thiacloprid (THI) metabolism by V. boronicumulans J1 involved hydrolysis of the N-cyanoimino group to form an N-carbamoylinino group containing metabolite, THI amide. Resting cells of V. boronicumulans J1 degraded 62.5% of the thiacloprid at a concentration of 200 mg/L in 60 h, and 98% of the reduced thiacloprid was converted to the final metabolite thiacloprid amide. A 2.6 kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an α-subunit of 213 amino acids and a β-subunit of 221 amino acids. The nitrile hydratase gene was recombined into plasmid pET28a and overexpressed in Escherichia coli BL21 (DE3). The resting cells of recombinant E. coli BL21 (DE3)-pET28a-NHase with overexpression of nitrile hydratase transformed thiacloprid to its amide metabolite, whereas resting cells of the control E. coli BL21 (DE3)-pET28a did not. Therefore, the major hydration pathway of thiacloprid is mediated by nitrile hydratase.