Background and aimsSoil nitrogen (N) transformation is a critical step in grassland N cycling. Nitrogen fertilization, a widely used restoration strategy in global grasslands, alters soil N transformation. Nevertheless, the responses of soil N transformation to multiple N input levels and the driving factors remain unclear.MethodsWith 15N tracing technique, we assessed the variations of gross and net N mineralization and nitrification rates across wide-ranging N fertilization levels (0, 2, 5, 10, and 20 g N m-2 yr-1) after seven years of treatment in a temperate grassland. Plant, soil and microbial traits were analyzed to explore the regulatory mechanisms of N fertilization on soil N transformations.ResultsBoth soil gross N mineralization (GNM) and nitrification rates (GN) showed positive responses to increasing N fertilization levels, with consequences on net N mineralization and nitrification rates. The consistent increases of GNM were primarily driven by the soil dissolved organic N availability. The enhancement of GN was attributed to the increases in GNM-derived NH+ content and the ammonium-mediated decreases in ammonia-oxidizing archaea to bacteria ratio (AOA:AOB). The role of GNM was more predominant under higher N fertilization rate.ConclusionMineralization-derived substrates quantity and ammonia-oxidizing community structure co-driven the responses of grassland soil nitrification to N fertilization, with soil substrates being predominant under higher N fertilization. Our findings emphasize that the drivers of soil N transformation varied among different N fertilization conditions, and therefore improve our mechanistic understanding of soil N turnover in a world with huge spatial variations of N enrichment.