<p>Legumes represent a crucial component of grassland nitrogen (N) cycling through symbiotic N₂ fixation (SNF). This study investigated the temporal dynamics of legume N acquisition under 6- and 12-year grazing exclusion regimes in alpine grasslands of the Qinghai-Tibetan Plateau using the ¹⁵N natural abundance technique. Analyses revealed a pronounced shift in N acquisition patterns: the amount of N derived from the atmosphere (Ndfa) in <i>Astragalus arnoldii</i> decreased from 1.00&#xa0;g m ^− 2 after 6 years of exclusion to 0.44&#xa0;g m ^− 2 after 12 years. Vegetation surveys indicated a marked decline in plant diversity, with the Shannon-Wiener index falling from 1.70 to 1.50, while the relative importance value of <i>A. arnoldii</i> was reduced from 0.22 to 0.17. Soil analyses demonstrated significant changes in the 20–30&#xa0;cm depth layer, where bulk density decreased from 1.76 to 1.42&#xa0;g cm ^− 3, soil water content increased from 5.42% to 9.85%, and total N concentration rose from 0.43 to 0.85&#xa0;g kg ^− 1 over the exclusion period. Structural equation modeling confirmed that grazing exclusion indirectly influenced legume N acquisition, primarily mediated through alterations in vegetation diversity and soil properties. These findings establish that exclusion duration critically regulates legume N acquisition, with short-term benefits being offset by long-term suppression through competitive exclusion and soil N accumulation. This study provides quantitative evidence to support the optimization of grazing exclusion strategies for sustaining legume N acquisition in alpine grassland ecosystems.</p>