Root litter decomposition is the dominant source of soil organic carbon (C) and nitrogen (N) in grasslands. Few studies, however, have explored the effect of root litter diversity on soil C and N cycling. This study investigated the effects of species diversity and functional traits of root litter on soil CO2 and N2O release, net ammonification, net nitrification, and net N mineralization based on a 56-day incubation of grassland soils with root litter mixtures containing one, two, or four native plant species. The increasing species richness of root litter decreased the cumulative CO2 and N2O release in the soil, but enhanced the net ammonification, nitrate immobilization, and N mineralization. Root litter diversity has a predominant non-additive antagonistic effect on the release of soil CO2 and N2O, and a synergistic effect on the net ammonification, nitrate immobilization, and N mineralization in the soil. The functional identity rather than functional diversity of root traits explains most of the variation in soil C and N cycling. A high C: N ratio and low concentrations of N, P, K, and Di-O-alkyl-C (characteristic of celluloses) were found to be key to the antagonistic effects associated with cumulative release of CO2 from the soil. For net N ammonification and mineralization, the synergistic effect was principally induced by the high levels of carbohydrate-C and N and the low C: N ratios in root litter mixtures. Our study highlights the role and mechanisms of increased root litter diversity in decreasing soil CO2 and N2O release and in increasing the net N mineralization via non-additive antagonistic and synergistic effects of dominant root traits.