Functional genes involved in nitrogen (N) cycling regulate soil nitrification, denitrification and N2O emissions. However, the general patterns and variability of N functional genes in response to N addition, and their association with N2O emission have not been synthesized for terrestrial ecosystems. We synthesized 2068 observations from 144 papers to explore the impact of N addition on the abundance and diversity of N functional genes, and their relationship to N2O emissions in croplands, grasslands and forests on a global scale. In croplands, N addition increased N2O emissions (109%), the abundance of ammonia-oxidizing archaea (AOA) (19%), ammonia-oxidizing bacteria (AOB) (95%), nirK (52%), nirS (40%) and nosZ (41%), and the diversity of AOB (15%), nirS (12%) and nosZ (11%). In grassland, N addition increased AOB abundance (130%) and decreased the abundance of nirS (−99%) and nosZ (−58%) genes, but in forests, significant effects were only found for the abundance of AOA (35%) and AOB (121%). N2O emission was negatively correlated with the abundance of nosZ, but positively correlated with the abundance of AOA and AOB. Apart from the abundance of functional AOA, AOB and nosZ genes, climate variables (precipitation and temperature), and available N concentrations were the main factors explaining the variation in N2O emission with N addition, as shown by random forest analysis. These findings indicate that impacts on N functional genes that encode enzymes involved in nitrification (AOA, AOB) and in the transformation of N2O to N2 (nosZ) are the main mechanisms behind the effect on N fertilizer-induced N2O emissions.