The optimal grazing hypothesis posits that moderate herbivore grazing can stimulate grassland productivity by increasing the turnover of soil nutrients and promoting plant compensatory growth. While this paradigm has been well-documented for aboveground biomass, its implications for soil organic carbon (SOC) dynamics — particularly the formation, chemical composition, and long-term stability of SOC - remain inadequately explored. Furthermore, in the context of widespread anthropogenic nutrient enrichment, it is unclear whether grazing-mediated nutrient turnover or synthetic fertilizer inputs more effectively enhance SOC sequestration and stability in grassland ecosystems. To address these uncertainties, a 17-year in situ experiment was conducted in an alpine meadow on the Tibetan Plateau, a critical carbon sink region sensitive to global change. The study manipulated grazing exclusion (moderate grazing) and nitrogen (N) addition treatments using distinct chemical forms (ammonium-N, nitrate-N, and their mixture) to investigate effects of grazing and fertilization on SOC dynamics. Soil samples from 0 —10 cm (surface) and 10 — 20 cm (subsurface) layers were analyzed for total SOC content, mineral-associated organic carbon (MAOC), and particulate organic carbon (POC) — key indicators of SOC stability. Advanced solid-state 13C nuclear magnetic resonance (13C -NMR) spectroscopy was utilized to characterize molecular-level changes in SOC functional groups, including alkyl-C (recalcitrant compounds), O-alkyl-C (labile carbohydrates), and carbonyl-C (oxidized components). Results demonstrated that prolonged grazing exclusion triggered substantial SOC depletion across both soil layers, with surface soil (0 — 10 cm) exhibiting a 17.8% reduction compared to winter grazed plots. While grazing exclusion elevated POC in the surface layer by 9.6%, it concurrently decreased MAOC — the mineral-stabilized carbon fraction with longer residence times — by 27.3%, indicating a net decline in both SOC quantity and stability. 13C-NMR analysis revealed that grazing exclusion altered SOC chemistry, reducing alkyl-C (from 24% to 22% of total SOC) while increasing O-alkyl-C (from 46% to 51%) in surface soils. This shifted the alkyl-C/O-alkyl-C ratio — a stability index — from 0.52 to 0.43, confirming SOC destabilization. Long-term N addition did not significantly alter SOC content compared with grazing exclusion. However, N form significantly influenced SOC composition and stability. Ammonium-N (NH4+) reduced surface MAOC by 3% relative to nitrate-N (NO3-), while increased POC by 5%, suggesting ammonium promoted labile carbon accumulation over mineral-associated stabilization. 13C-NMR data corroborated this: NH4+ decreased alkyl-C (11%) and carbonyl-C (10%), while elevated O-alky-C (7%) in surface soils, whereas NO3- induced milder changes. This divergence likely stems from ammonium’s stronger acidification effect (pH decline by 0.42 units under NH4+ vs. 0.18 under NO3-), which inhibits mineral-organic binding and accelerates decomposition of stabilized carbon. Notably, subsurface SOC (10 — 20 cm) dynamics showed positive correlations with POC. Mechanistically, ammonium addition acidified soils, pH shifts modulated the organo-mineral associations critical for MAOC formation, explaining SOC variability. The study highlights that anthropogenic N inputs, particularly ammonium-based fertilizers, may inadvertently undermine SOC stability. Our findings suggest that balance grazing intensity with pH-conscious nutrient amendments to optimize both productivity and carbon persistence in alpine ecosystems.