Quantification of functional genes involved in nitrogen (N) transformation improves our understanding of N-cycling microbial population responses to environmental disturbance. Agricultural N fertilization affects N-cycling gene abundances in soil, but the general patterns and variability of N cycling gene abundances in response to N fertilization have yet to be synthesized. We conducted a meta-analysis comprising 47 field studies in agricultural ecosystems. We included five marker genes important to N-cycling: nifH (encoding nitrogenase; key enzyme for N fixation), amoA (encoding ammonia monooxygenase; key enzyme for nitrification), nirK and nirS (encoding nitrite reductase; key enzyme for denitrification), and nosZ (encoding nitrous oxide reductase; key enzyme for denitrification). We found that N fertilization had no effect on the abundance of nifH, but significantly increased archaeal amoA (31%), bacterial amoA (313%), nirK (53%), nirS (40%) and nosZ (75%), respectively. N fertilizer form (inorganic versus organic) strongly affected the response of most selected N-cycling genes to N fertilization; organic fertilizers often had a much stronger effect than inorganic fertilizers. N fertilization duration, crop rotation, and soil pH were also important factors regulating the response of most N-cycling genes to N fertilization. Genes involved in nitrification and denitrification were significantly correlated with each other. Improvement in understanding of the response of N-cycling gene abundance to enhanced N input will help develop quantitative models of N availability and N fluxes and improve strategies for reducing reactive N gas emissions and N management in agricultural ecosystems.