The community structure and abundance of nirK-type denitrifying bacteria in different soil layers (0-20 cm and 20-40 cm) under various fertilization regimes in Wuwei, Gansu Province were investigated by the combination of terminal restriction fragment length polymorphism (T-RFLP) and real-time quantitative PCR. Results showed that the nirK-type denitrifying bacteria community structure was significantly affected by fertilization regimes, especially for 70, 156 and 190 bp T-RFs that represented the dominant populations in greenhouse soil. Fertilization regimes significantly influenced the abundance of nirK gene in the 0-20 cm soil layer with the highest abundance of nirK gene copy number (2.16 x 10(7) copies x g(-1) soil) detected in the manure treatment (M), which was 2.04 and 2.02 times of that in the control (CK) and chemical fertilizer (NPK) treatments, respectively. Both the dominant population and abundance of nirK-type denitrifying bacteria in the greenhouse soil were significantly different between the 0-20 cm and 20-40 cm soil layers, and the nirK-type denitrifying bacteria community structure and abundance in the greenhouse soil were obviously different from that in the field. Soil pH, soil organic matter content and nitrate-N content had the greatest influence on the bacterial community composition. Phylogenetic analysis indicated that there were not only anaerobic nirK-type denitrifying bacteria in greenhouse soil, but also aerobic denitrifying bacteria, such as Rhizobium, Ochrobactrum, Agrobacterium.