Background and aims <p>Soil 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.</p> Methods <p>With 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.</p> Results <p>Both 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.</p> Conclusion <p>Mineralization-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.</p>