The composition of culture-independent microbial communities and the change of nitrogenase activities under the application of butachlor in paddy soil were investigated. Nitrogen-fixation ability was expressed by the amount of acetylene reduction, and changes of microbial communities were studied by using denaturing gradient gel electrophoresis (DGGE) technique; afterward, minimum distance (MD, in brief) statistics was applied to determine the cluster numbers in UPGMA dendrograms. The results showed that the reduction of acetylene was suppressed shortly after butachlor application but was augmented after 37 days in both upper and lower layer soils. From UPGMA dendrograms, the diazotrophic divergences ranged from 33% to 64% throughout rice growth stages. For general bacterial communities, the diversities ranged from 28% to 52%. The divergences became higher with the cultivation period, and the application of butachlor imposed a significant variation on microbial community shift, which may be a reason for the boosting nitrogen-fixation ability in paddy soils.