Nitrogen (N) deposition is increasing at unprecedentedly rate due to anthropogenic activities. The N deposition is altering the microbial activity and coupled C and N cycling through changing soil pH, N and carbon (C) availability. (1) N-related functional genes (NFGs) (N fixation, nifH; N mineralization, chiA; nitrification, AOA- and AOB-amoA；denitrification，narG，nirK，nirS and nosZ) regulate the main N transformation processes. However, empirically simulated N deposition experiments have been exclusively conducted through large and infrequent N fertilization, which may have caused contrasting effects on N cycling, compared with small and frequent N additions which were close to natural N deposition. Independently manipulated different rates (0-50 g N m–2yr–1) and frequencies (2 and 12 times per year) were selected to quantify the NFG abundances in Xilingol grassland, Inner Mongolia. The nifH, AOA-amoA, nirS, and nosZ gene abundances increased due to improved available N at low N rates, but were suppressed by salt toxicity and acidification at high N rates. The significant lower abundances of nifH, nirS, and nosZ were detected under the infrequent N addition plots due to stronger suppression by soil acidification and salt toxicity. The AOB-amoA gene abundance increased with increasing N rates and had higher abundance in the infrequent plots due to the higher NH4+-N concentration. The results demonstrated that small and frequent N addition should be employed to project the effects of N deposition on microbial functional groups as well as on N transformations.