Growing evidence suggests that tropospheric ozone has widespread effects on vegetation, which can contribute to alter ecosystem carbon (C) dynamics and belowground processes. In this study, we used intact soil mesocosms from a semi-improved grassland and investigated the effects of elevated ozone, alone and in combination with nitrogen (N) fertilization on soil-borne greenhouse gas emissions and ecosystem C fluxes. Ozone exposure under fully open-air field conditions was occurred during the growing season. Across a one-year period, soil methane (CH4) and nitrous oxide (N2O) emissions did not differ between treatments, but elevated ozone significantly depressed soil CH4 uptake by 14% during the growing season irrespective of N fertilization. Elevated ozone resulted in a 15% reduction of net ecosystem exchange of carbon dioxide, while N fertilization significantly increased ecosystem respiration during the growing season. Aboveground biomass was unaffected by elevated ozone during the growing season but significantly decreased by 17% during the non-growing season. At the end of the experiment, soil mineral N content, net N mineralization and extracellular enzyme activities (i.e., cellobiohydrolase and leucine aminopeptidase) were higher under elevated ozone than ambient ozone. The short-term effect of single application of N fertilizer was primarily responsible for the lack of the interaction between elevated ozone and N fertilization. Therefore, results of our short-term study suggest that ozone exposure may have negative impacts on soil CH4 uptake and C sequestration and contribute to accelerated rates of soil N-cycling.