Aims <p>Glomalin-related soil proteins (GRSP) and amino sugars are key components of soil organic carbon (SOC) and are significantly influenced by nitrogen (N) enrichment. Many regions worldwide are experiencing N deposition cessation due to greenhouse gas emission policies. As a global grassland management practice, mowing impacts soil carbon (C) and nutrient cycling. The effects of N cessation and mowing on GRSP, amino sugars, and their contributions to SOC remain unclear.</p> Methods <p>We investigated the individual and interactive effects of a long-term field experiment involving N cessation and mowing treatments on GRSP and amino sugars and their relative contributions to SOC in a temperate grassland.</p> Results <p>Both GRSP and amino sugars, along with their relative contributions to SOC, decreased with increasing historical N addition rates and without mowing. This was attributed to persistent effects of historical N addition on soil microbial biomass and acidification, which suppressed GRSP and amino sugars. However, as indicated by the resource stoichiometry and extracellular enzyme activity, N cessation and mowing interactively induced stronger nutrient limitation, exerting diverged effects on GRSP and amino sugars. Specifically, nutrient limitation increased GRSP by 24.15%, while amino sugars increased by 3.57%, altering their relative contributions to SOC.</p> Conclusions <p>Our findings suggest that GRSP is relatively more stable and contributes more to SOC than amino sugars after N cessation, especially under mowing. This study enhances understanding of soil C dynamics in grasslands after N cessation. Results provide a decision-making basis for the adaptive management of grassland ecosystems under the “dual C” strategy goals.</p>