In order to understand the effect of organic manure application on the structure of denitrifying bacteria in red soil, four treatments were designed and compared:1) CK, no manure; 2) LM, low organic manure; 3) ML, high organic manure + lime; and 4) HM, high amount organic manure. The diversity of the nirK gene was estimated by terminal restriction fragment length polymorphism (T-RFLP) and DNA sequencing of the cloned library. The results showed that the 288 clones selected in each treatment can be divided into 78 clusters, and the dominant groups in each nirK library were of the same cluster, accounting for 51% of the CK, while the other three treatments were 33%, 32% and 27%, respectively. The similarity between the four libraries was between 37.50% and 45.34%. The phylogenetic tre e analysis of the 51 OTUs showed that 6 OTUs had the highest similarity with Ochrobactrum sp., accounting for 11.8% of the total number of sequencing. The remaining 45 OTUs belonged to the uncultured cluster, accounting for 88.2% of the total number of sequencing. Organic manure addition increased the diversity of the nirK gene, but some types of denitrifying bacteria didn't appear in the original red soil environment.