Whether and how CO₂ and nitrogen (N) availability interact to influence carbon (C) cycling processes such as soil respiration remains a question of considerable uncertainty in projecting future C-climate feedbacks, which are strongly influenced by multiple global change drivers, including elevated atmospheric CO₂ concentrations (eCO₂) and increased N deposition. However, because decades of research on the responses of ecosystems to eCO₂ and N enrichment have been done largely independently, their interactive effects on soil respiratory CO₂ efflux remain unresolved. Here, we show that in a multifactor free-air CO₂ enrichment experiment, BioCON (Biodiversity, CO₂, and N deposition) in Minnesota, the positive response of soil respiration to eCO₂ gradually strengthened at ambient (low) N supply but not enriched (high) N supply for the 12-y experimental period from 1998 to 2009. In contrast to earlier years, eCO₂ stimulated soil respiration twice as much at low than at high N supply from 2006 to 2009. In parallel, microbial C degradation genes were significantly boosted by eCO₂ at low but not high N supply. Incorporating those functional genes into a coupled C-N ecosystem model reduced model parameter uncertainty and improved the projections of the effects of different CO₂ and N levels on soil respiration. If our observed results generalize to other ecosystems, they imply widely positive effects of eCO₂ on soil respiration even in infertile systems.