Abstract Objective: This study was designed to reveal the potential molecular mechanisms of long-term overgrazing-induced dwarf of sheepgrass (Leymus chinensis). Methods: The electrospray ionization-mass spectrometry (ESI-MS) system was used to generate the proteomic data in dwarf sheepgrass from long-term overgrazed rangeland and normal sheepgrass from long-term enclosed rangeland. The differentially expressed proteins (DEPs) between dwarf and normal sheepgrass were identified, following the analyses of potential functions and interactions of DEPs. Additionally, the expressions of DEPs were confirmed by high performance liquid chromatography-mass spectrum (HPLC-MS) system with multiple reaction monitoring (MRM) method. Results: A total of 51 up-regulated proteins and 53 down-regulated proteins were identified in the dwarf sheepgrass, compared with the normal controls. Some proteins like SAT5_ARATH and DAPA_MAIZE were enriched in the pathway of amino acid biosynthesis. In the protein-protein interaction network, RPOB2_LEPTE, A0A023H9M8_9STRA, ATPB_DIOEL, RBL_AMOTI and DNAK_GRATL interacted with each other. Furthermore, 4 modules were extracted from the PPI network. In addition, the HPLC-MS analysis confirmed the up-regulation of ATPB_DIOEL and the down-regulation of DNAK_GRATL in dwarf samples compared with the controls. Conclusion: The up-regulated ATPB_DIOEL and down-regulated DNAK_GRATL, as well as the proteins that interacted with them, such as RPOB2_LEPTE, A0A023H9M8_9STRA and RBL_AMOTI, may be associated with the long-term overgrazing-induced sheepgrass dwarf.