Predicting if ecosystems will mitigate or exacerbate rising CO 2 requires understanding how elevated CO 2 will interact with coincident changes in diversity and nitrogen (N) availability to affect ecosystem carbon (C) storage. Yet achieving such understanding has been hampered by the difficulty of quantifying belowground C pools and fluxes. Thus, we used mass balance calculations to quantify the effects of diversity, CO 2 , and N on both the total amount of C allocated belowground by plants (total belowground C allocation, TBCA) and ecosystem C storage in a periodically burned, 8-year Minnesota grassland biodiversity, CO 2 , and N experiment (BioCON). Annual TBCA increased in response to elevated CO 2 , enriched N, and increasing diversity. TBCA was positively related to standing root biomass. After removing the influence of root biomass, the effect of elevated CO 2 remained positive, suggesting additional drivers of TBCA apart from those that maintain high root biomass. Removing root biomass effects resulted in the effects of N and diversity becoming neutral or negative (depending on year), suggesting that the positive effects of diversity and N on TBCA were related to treatmentdriven differences in root biomass. Greater litter production in high diversity, elevated CO 2 , and enhanced N treatments increased annual ecosystem C loss in fire years and C gain in non-fire years, resulting in overall neutral C storage rates. Our results suggest that frequently burned grasslands are unlikely to exhibit enhanced C sequestration with increasing atmospheric CO 2 levels or N deposition.