Deeper understanding of the link with soil microbial diversity and their function is needed for the productivity and stability of agricultural ecosystems. Intensive agriculture systems that repeatedly apply nitrogen (N) are reported to change the soil microbial community and N-cycling functions, with increases in the abundances of ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, it was not clear how the diversity of soil microbes were related to the changes in the abundance of AOA and AOB after repeated N application. Thus, the aim of the present study was to elucidate how microbial diversity is linked to the ammonium oxidation at the repeated N application phase. To produce a diversity gradient, serially diluted soil suspension (4 level; 10−1, 10−2, 10−3, 10−4) was inoculated to the sterilized soil. After the 10 weeks incubation and repeated application of  (NH₄)₂SO₄, quantitative abundances of AOA and AOB, and community were analyzed. With N application, the abundance of AOB increased most rapidly in 10−1 while an increase in AOA was observed in 10−4. The results suggest diversity gradient was the important factor controlling the abundance of AOA and AOB. Further, in soil with relatively lower microbial diversity, AOA might play more important role for nitrification.