Although it is known that multiple interactions among soil abiotic properties, plant functional traits and soil microbial properties influence nutrient cycling, the relative contribution of each of these groups of variables is poorly understood. We manipulated grassland plant functional composition and soil nitrogen (N) availability in a multi-site mesocosm experiment to quantify their relative effects on soil N turnover. Overall, root traits, denitrification potential, as well as N and water availability, best explained variation in measured ecosystems properties, especially the trade-off between nutrient sequestration and plant biomass production. Their relative contributions varied with soil inorganic N availability. In relatively N-poor soils, N turnover was mainly controlled by microbial and soil abiotic properties, whereas in the relatively N-richer soils, it was mainly controlled by plant functional traits. This experiment is a strong demonstration of the role of functional characteristics of both plants and soil microbes, and their interplay with soil N availability, as biotic markers of N turnover in grasslands.