Long-term nitrogen fertilization imposes strong selection on nitrifying communities in agricultural soil, but it remains poorly understood how a progressively changing niche will affect potentially active nitrifiers in field. Using a 44-year-old grassland fertilization experiment, we investigated community shift of active nitrifiers by DNA-based stable isotope probing (SIP) of field soils that received no fertilization (CK), high levels of organic cattle manure (HC) and chemical N fertilization (CF). Incubation of DNA-SIP microcosms showed significant nitrification activities in CF and HC soils, whereas no activity occurred in CK soils. 44 years of inorganic N fertilization selected for only 13C-AOB, whereas cattle slurry applications created a niche in which both AOA and AOB could be actively 13C-labeled. Phylogenetic analysis indicated that Nitrosospira sp. 62-like AOB dominated inorganically fertilized CF soils, and Nitrosospira sp. 41-like AOB were abundant in organically fertilized HC soils. 13C-AOA in HC soil were affiliated with the 29i4 lineage. 13C-NOB were dominated by both Nitrobacter- and Nitrospira-like communities in CF soils, whereas the latter was overwhelmingly abundant in HC soil. The 13C-labeled nitrifying communities in SIP microcosms of CF and HC soils were largely similar to those predominant under field conditions. These results provide direct evidence for strong selection of distinctly active nitrifiers after 44 years of different fertilization regimes in the field. Our finding implies that niche differentiation of nitrifying communities could be assessed as a net result of microbial adaption over 44 years to inorganic and organic N fertilizations in the field, and distinct nitrifiers have been shaped by intensified anthropogenic N input.