AbstractWe used a seven-year urea gradient applied field experiment to investigate the effects of nitrogen (N) applications on soil N hydrolytic enzyme activity and ammonia-oxidizing microbial abundance in a typical steppe ecosystem in Inner Mongolia. The results showed that N additions inhibited the soil N-related hydrolytic enzyme activities, especially in 392 kg N ha⁻¹ yr⁻¹ treatment. As N additions increased, the amoA gene copy ratios of ammonia-oxidizing archaea (AOA) to ammonia-oxidizing bacteria (AOB) decreased from 1.13 to 0.65. Pearson correlation analysis showed that the AOA gene copies were negatively related with NH₄⁺-N content. However, the AOB gene copies were positively correlated with NO₃⁻-N content. Moderate N application rates (56–224 kg N ha⁻¹ yr⁻¹) accompanied by P additions are beneficial to maintaining the abundance of AOB, as opposed to the inhibition of highest N application rate (392 kg N ha⁻¹ yr⁻¹) on the abundance of AOB. This study suggests that the abundance of AOB and AOA would not decrease unless N applications exceed 224 kg N ha⁻¹ yr⁻¹ in temperate grasslands in Inner Mongolia.