Abstract
<title>Abstract</title> <p> Grazing alters the interactions between plants and microbial and has a significant impact on grassland ecosystems, with root exudates (RE) serving as the primary mechanism by which plants regulate and maintain microbial communities. However, little is known about how RE regulate microbial communities under grazing stress, or whether the efficacy of key metabolites is limited by their thermodynamic properties or molecular structure. By analyzing RE metabolites, rhizosphere microbial communities, and the physicochemical properties of rhizosphere soil in typical grassland in Inner Mongolia, China, we found that: (1) short-term grazing exerted a stronger effect on RE metabolites of <italic>Stipa grandis</italic> . RE metabolites explained significantly more variation in rhizosphere microbial communities than soil physicochemical properties, and showed deeper coupling with rhizosphere fungal communities; (2) the abundance of RE metabolites varied with grazing duration and intensity. Under short-term grazing pressure, <italic>S. grandis</italic> upregulated energy-rich lipids and specific signaling molecules (e.g., (-)-jasmonic acid) to drive rapid bacterial growth. In contrast, under long-term moderate grazing, plants shifted toward sustained secretion of defensive secondary metabolites, maintaining mutually beneficial associations with rhizosphere fungi; (3) under resource-limited conditions induced by long-term moderate grazing, the topological position of key RE metabolites in bacteria-associated networks were constrained by thermodynamic properties, whereas their position in fungi-associated networks was generally constrained by molecular structural complexity across multiple grazing treatments. These findings fill a critical knowledge gap in grassland ecosystem research and provide an important theoretical basis, from a microbiological perspective, for enhancing grassland productivity and the restoration of degraded grasslands more broadly. </p>