Toxic effects of two agrochemicals on nifH gene in agricultural black soil were investigated using denaturing gradient gel electrophoresis (DGGE) and sequencing approaches in a microcosm experiment. Changes of soil nifH gene diversity and composition were examined following the application of acetochlor, methamidophos and their combination. Acetochlor reduced the nifH gene diversity (both in gene richness and diversity index values) and caused changes in the nifH gene composition. The effects of acetochlor on nifH gene were strengthened as the concentration of acetochlor increased. Cluster analysis of DGGE banding patts showed that nifH gene composition which had been affected by low concentration of acetochlor (50 mg/kg) recovered firstly. Methamidophos reduced nifH gene diversity at the first week while the high concentration of methamidophos (250 mg/kg) produced prominent effects on nifH gene diversity in the following weeks. Cluster analysis showed that minimal changes of nifH gene composition were found at 1 week and maximal changes at 4 weeks. Toxic effects of acetochlor and methamidophos combination on nifH gene were also apparent. Different nifH genes (bands) responded differently to the impact of agrochemicals: four individual bands were eliminated by the application of the agrochemicals, five bands became predominant by the stimulation of the agrochemicals, and four bands showed strong resistance to the influence of the agrochemicals. Fifteen prominent bands were partially sequenced, yielding 15 different nifH sequences, which were used for phylogenetic reconstructions. All sequences were affiliated with the alpha-and beta-proteobacteria, showing higher similarity to eight different diazotrophic genera.