Root exudate profile is expected to be altered by global change drivers, with significant implications for plant nutrition. Exposure to elevated atmospheric carbon dioxide (eCO2) increases the quantity and alters the quality of exudates, which likely affects microbial activity and nitrogen (N) cycling. However, it is uncertain whether such changes will result in greater N availability for plants. In this field experiment, we used an automated root exudation system in a forest soil to mimic the increase in exudate C:N ratio observed under eCO2. After six months of continuous application, we measured N transformation rates in O-horizon soils and in root and fungi exclusion soil bags (41 μm and 1 μm mesh sizes) to partition the role of fungi and bacteria. Increasing exudate C:N ratio stimulated gross N mineralization, especially in the rhizosphere, by shifting microbial nutrient acquisition strategy towards a N-mining strategy. High exudate C:N ratio increased nitrification in the absence of roots when both fungi and bacteria were present. These results demonstrate that N transformations are driven more by the C:N stoichiometry than by labile C alone in root exudates, and are largely influenced by the rhizosphere environment. Exudate stoichiometry thus may play a key role in alleviating N limitation under future atmospheric CO2 concentration.