Purpose Microbial processes driving nitrogen (N) cycling are hot topics in terms of increasing N deposition. Abundances of N-related functional genes (NFG) can be most responsive to N deposition and commonly used to represent N transformation rates. However, empirically simulated N deposition has been exclusively conducted through large and infrequent N fertilization, which may have caused contrasting effects on NFGs. Therefore, experiments with small and frequent N additions closed to natural deposition are necessary. Materials and methods Independently manipulated N addition rates (i.e., 0, 5, 10, 15, 20, and 50 g N m-2 year-1) and two frequencies (2 times per year addition as conventional large and infrequent N fertilization (2 N), and 12 times per year addition simulating small and frequent N deposition (12 N), respectively) were conducted in a long-term field experiment of a semiarid grassland in northern China. Quantification analysis using real-time PCR were carried out for NFGs, including nifH for N fixation, chiA for N mineralization, archaeal (AOA) and bacterial (AOB) amoA for nitrification, and narG, nirS, nirK, and nosZ for denitrification. Results and discussion NFG abundances showed distinct sensitivities to N addition rates. The nifH, AOA-amoA, nirS, and nosZ gene abundances increased due to improved available N at low N rates, but suppressed by salt toxicity and acidification at high N rates. Large changes of chiA and AOB-amoA gene abundances highlighted their great sensitivities to the N enrichment. The abundance of AOB-amoA was more sensitive to N addition than AOA-amoA, but AOA-amoA dominated in absolute numbers and they predominated the ammonia-oxidation under different conditions. The N addition frequencies caused significant lower gene abundances of nifH, nirS, and nosZ under the 2-N frequency due to stronger suppression of acidification and salt toxicity and resulted in significant higher AOB-amoA gene abundances in response to higher N availability under the 2-N frequency. Conclusions The NFGs abundances responded to N addition rates distinctly, highlighting that the driven processes involved in N cycling were altered by the N addition rates. The different effects of two N addition frequencies on NFG abundances demonstrated that conventional large and infrequent N fertilization cannot represent N deposition, and 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.