[Objective] Ammonia oxidation is the limited step of nitrification and it is the main microbial pathway of N₂O emissions in aerobic soils. Grassland soils are sinks for CH₄and sources of N₂O. However, the mechanisms of the interactions between methane and ammonia oxidation under various N levels in grassland soils remain elusive.[Method] A microcosm experiment was implemented using Inner Mongolia grassland soils based on DNA-Stable-Isotope-Probing (DNA-SIP), and the methane and ammonia oxidation under various N levels was studied.[Result] The results showed that methane-oxidizing activity was stimulated by low N while it was suppressed by high N application. Contrarily, the nitrification activity was stimulated by both low and high N addition. The Methylobacter lineage of methane-oxidizing bacteria(MOB) were the active players in methane oxidation, while nitrifying communities dominated by Nitrosospira lineage of AOB and Nitrospira-like nitrite-oxidizing bacteria (NOB) were the active players in nitrification. Also, the network analysis unraveled significant negative relationships between active Methylobacter MOB and Nitrosospira AOB/Nitrospira NOB, which suggested competitive interactions between active nitrifiers and methanotrophs in grassland soils.[Conclusion] The interactions between active methanotrophs and nitrifiers can be affected by N levels. Our results provide theoretical support for regulating CH₄ sinks and N₂O sources of grassland soils and for greenhouse gas mitigation.