Nitrogen fertilizer can affect the abundance and composition of soil ammonia-oxidizing bacteria(AOB)and ammonia-oxidizing archaea(AOA).This study investigated the response of AOB and AOA to different application years(4 years，4Y；17 years，17Y；32 years，32Y； 0 year，0Y)of nitrogen fertilizer using real-time polymerase chain reaction(RT-PCR)and denaturing gradient gel electrophoresis(PCR-DGGE).The results showed that long-term nitrogen fertilizer application resulted in soil acidification，and the pH was decreased by 0.88 in 32Y soil compared with 4Y soil.The 4Y soil had the highest copy numbers of AOB amoA genes among the treatments，whereas those of AOA amoA genes were found in 32Y soil.The bacterial amoA gene copy numbers ranging from 6.46 × 105 to 8.32 × 107 per gram of dry soil were significantly higher than those of archaeal amoA genes，ranging from 1.70 × 104 to 1.20 × 105 per gram of dry soil.The AOB population size was positively correlated with the potential nitrification rates(PNR)，which indicated a potential role of AOB in nitrification. No significant correlations were observed among the population size of AOA，soil pH and PNR.DGGE patterns revealed that different application years of nitrogen fertilizer resulted in an obvious change of the AOB community composition，but not of AOA.These results suggest that long-term application of nitrogen fertilization had a significant impact on AOB abundance and composition，while minimal on AOA abundance in mulberry field soil.