Rising atmospheric CO concentrations are expected to increase nitrous oxide (N O) emissions from soils via changes in microbial nitrogen (N) transformations. Several studies have shown that N O emission increases under elevated atmospheric CO (eCO ), but the underlying processes are not yet fully understood. Here, we present results showing changes in soil N transformation dynamics from the Giessen Free Air CO Enrichment (GiFACE): a permanent grassland that has been exposed to eCO , +20% relative to ambient concentrations (aCO ), for 15 years. We applied in the field an ammonium-nitrate fertilizer solution, in which either ammonium (NH4+) or nitrate (NO3-) was labelled with N. The simultaneous gross N transformation rates were analysed with a N tracing model and a solver method. The results confirmed that after 15 years of eCO the N O emissions under eCO were still more than twofold higher than under aCO . The tracing model results indicated that plant uptake of NH4+ did not differ between treatments, but uptake of NO3- was significantly reduced under eCO . However, the NH4+ and NO3- availability increased slightly under eCO . The N O isotopic signature indicated that under eCO the sources of the additional emissions, 8,407 μg N O-N/m during the first 58 days after labelling, were associated with NO3- reduction (+2.0%), NH4+ oxidation (+11.1%) and organic N oxidation (+86.9%). We presume that increased plant growth and root exudation under eCO provided an additional source of bioavailable supply of energy that triggered as a priming effect the stimulation of microbial soil organic matter (SOM) mineralization and fostered the activity of the bacterial nitrite reductase. The resulting increase in incomplete denitrification and therefore an increased N O:N emission ratio, explains the doubling of N O emissions. If this occurs over a wide area of grasslands in the future, this positive feedback reaction may significantly accelerate climate change.