Polysaccharides from Glycyrrhiza are known to have several bioactive effects. Current studies on polysaccharides mainly focused on the biological activity of mixed components or large molecular weight polysaccharides. However, the structure-activity relationship between polysaccharides and immune regulation is still unclear. In this work, a low molecular weight GP1 (6.5 kDa) mainly composed of glucose was purified. Its backbone was determined as →4)-α-D-Glcp-(1→, →6)-α-D-Glcp-(1→ (9.3%), →4,6)-α-D-Glcp-(1→ and α-D-Glcp-(1→ residues. This structure gives GP1 the good antioxidant ability (91.85%) and phagocytic activity (129.15%) of RAW264.7 cell. Through in-vitro simulation, it was found that GP1 could be degraded by intestinal flora, increase the production of SCFAs by 83.44%, and promote the proliferation of beneficial bacteria. Further studies showed that GP1 effectively increased body weight, immune organ index, and intestinal barrier function of immunosuppressed mice. And GP1 also promoted the proliferation of goblet cells, CD4+ and CD8+ T lymphocytes by 31.40%, 20.61% and 50.15%, respectively. Meanwhile, GP1 also activated the expression ofTLR2, TLR4 and TLR6 gene in the TLR signaling pathway on the immune cell surface, thereby increasing the release of cytokines. These results indicated that the immunomodulatory of GP1 was closely related to intestinal flora, immune cell and TLR genes up-regulation.