Human-induced increases in nitrogen (N) deposition are common across many terrestrial ecosystems worldwide. Greater N availability not only reduces biological diversity but also affects the biogeochemical coupling of carbon (C) and N cycles in soil ecosystems. Soils are the largest active terrestrial C pool, and N deposition effects on soil C sequestration or release could have global importance. Here we show that 27-years of chronic N additions to prairie grasslands increased C sequestration in mineral soils and that a potential mechanism responsible for this C accrual was an N-induced increase in root mass. Greater soil C sequestration followed a dramatic shift in plant community composition from native species-rich C4-grasslands to naturalized species-poor C3-grasslands, which despite lower soil C gains per unit of N added, still acted as soil C sinks. Since both high plant diversity and elevated N deposition may increase soil C sequestration, but N deposition also decreases plant diversity, more research is needed to address the long-term implications for soil C storage of these two factors. Finally, because exotic C3 grasses often come to dominate N-enriched grasslands, it is important to determine if such N-dependent soil C sequestration occurs across C3-grasslands in other regions worldwide.