Abstract: Studies about ammonia oxidizing bacteria (AOB) have mainly focused on the topsoil and little has remained known about community distribution in the subsoil. There therefore has remained the need to understand the impact of long-term fertilization on AOB abundance, community structure and vertical distribution in order to deepen the exploration of microbial mechanisms of nitrogen (N) transformation and to develop sound fertilization regimes for sustainable soil quality in the study area and beyond. Thus, a long-term (1987-2015) fertilization experiment was set up in the brown earth in Shenyang Agriculture University, Liaoning Province, China. Four treatments were set, no fertilizer (CK), low N fertilizer (N₂), high N fertilizer (N₄) and low N fertilizer plus organic mature (M₂N₂). Soil samples were collected at three different depths (0-20 cm, 20-40 cm and 40-60 cm). The soil physico-chemical properties, 16S rDNA and AOB-amoA gene abundance (real-time PCR, qPCR) and AOB community structure and diversity (denaturing gradient gel electrophoresis, PCR-DGGE) were investigated. While soil pH decreased, the content of soil ammonium N (NH₄⁺-N) increased by 70.5%-939.21% and that of nitrate N (NO₃^--N) by 253.20%-625.48% in the fertilization treatments over CK treatment. Also while soil pH increased, the contents of soil NH₄⁺-N and NO₃^--N decreased with increasing soil depth, except for N₂ treatment. The results of qPCR showed that fertilization treatments increased AOB abundance, but decreased total bacterial abundance compared to CK treatment. AOB amoA gene abundance was generally higher at the 0-20 cm than at the 20-40 cm and 40-60 cm soil layers. AOB abundance peaked in the N₄ treatment, with 9.65×10⁵ copies per g dry soil. The Shannon diversity (H), evenness (E_H) and richness (S) indexes of AOB from DGGE fingerprints responded increasingly significantly (P < 0.05) to fertilization regimes and soil-fertilization interactions with increasing soil depth. Although the tested diversity indexes were highest in the surface soil (0-20 cm), N fertilizer treatments (N₂, N₄ and M₂N₂) significantly reduced AOB diversity indexes. Based on cluster analysis of the DGGE fingerprints, AOB community structure in the soil varied with fertilization treatments and soil depth. Three soil depths of high N fertilizer (N₄) treatment was grouped together clearly. For other treatments, it was grouped according to soil depth with no discernible difference in AOB community structure among CK, N₂ and M₂N₂ treatments. The 0-20 cm and 20-40 cm deep soils under fertilizer treatments formed single cluster with no less than 57% similarity, while 40-60 cm soil layer formed another cluster. Redundant gradient analysis (RDA) further showed that NO₃^--N (P=0.027) was the key factor that shaped AOB community under different fertilization treatments. The results indicated that AOB number and community structure diversity after long-term fertilization significantly varied with fertilization treatment, and showed obvious vertical distribution characteristics. Compared with chemical fertilizer (N₂ and N₄) application, organic manure plus chemical fertilizer (M₂N₂) more favorably improved soil pH and maintained AOB community diversity in the subsoil.