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High levels of N fertilization and acid deposition could cause soil acidification directly and indirectly. The nitrogen cycle, especially nitrification, makes a great contribution to the acidification of agricultural soils across China, which further leads to the mobilization of potentially toxic metals such as aluminum (Al) and manganese (Mn) and decerases crop yields. Chemicals (e.g., CaO) are amended as soil conditioners to relieve soil acidification. Ammonia oxidation, the rate-limiting step in the nitrification process, is driven by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Increasing evidence demonstrates that pH is one of the most important factors determining the niche separation of AOA and AOB, and AOA play the more important role in nitrification of acidic soils. However, abundant AOB have been detected in acidic soils but little is known about their ecological function. In this study, the effects of long-term N fertilization practices and liming on nitrification and ammonia oxidizers in acidic soils were investigated using quantitative PCR and DGGE methods combined with soil physiochemical analysis. Compared with a previous study conducted 6 years ago at the same site, N fertilizer application without liming further decreased soil pH (3.35—3.47) and potential nitrification rate (PNR) (0.02—0.14 µg NO-2 -N g-1 soil h-1), while 2 years liming alleviated soil acidification (pH 4.10—4.46) and increased PNR (0.22—0.34 µg NO-2 -N g-1 soil h-1) significantly. There was a significantly positive correlation between soil pH and PNR, indicating the increase in soil pH via liming had positive effects on nitrification in acidic soils. AOA amoA gene copy numbers (7.40 ×107—4.08 ×108 copies/ g) were significantly higher than their counterpart AOB (1.67 ×106—2.57 ×107 copies/ g) in soils that received different chemical N fertilizers. Ratios of AOA and AOB amoA gene abundance ranged between 10.9 and 44.3. After amending lime for two years in the N fertilizers treatment plots, AOA amoA gene copy numbers (8.23 ×106—4.89 ×107 copies/ g) decreased to a certain degree but AOB amoA gene copy numbers (1.99 ×107—5.42 ×107 copies/ g) increased significantly compared to their corresponding fertilization treatments without liming,resulting in lower ratios of AOA to AOB amoA gene abundance (0.83—1.94). Moreover, liming showed no clear influence on AOA community structure, but significantly increased AOB community diversity. Taken together, these results indicate that although the activity and abundance of AOA was generally greater than those of AOB in acidic soils, both AOA and AOB actively responded to disturbance in acidic soils based on their niche differentiation and had different effects on nitrification of acidic soils.