Polysaccharide of Tetrastigma hemsleyanum (THP) exert antioxidant, antibacterial, lipid-lowering, and anti-inflammatory properties, especially some evidences have highlighted the efficiency of it as an anti-tumor agent. However, as a biological macromolecule with bidirectional immune regulation, the immunological enhancement effects of THP on macrophages and its underlying mechanisms are still largely unknown. In the present study, THP was prepared and characterized, and then the effect of THP on Raw264.7 cell activation was investigated. Structural characteristics of THP showed that the average molecular weight was 370.26 kDa, and the main monosaccharide composition was galactose, glucuronic acid, mannose, and glucose at a ratio of 31.56: 25.15: 19.44: 12.60, with high viscosity causing by relative high uronic acid. For immunomodulatory activity investigation, THP promoted the production of NO, IL-6 and TNF-α, as well as the expression of IL-1β, MCP-1, iNOS and COX-2, which were almost completely inhibited by TLR4 antagonist. Further study showed that THP could activate NF-κB and MAPK signaling pathways, and thus enhanced the phagocytic activity of Raw264.7 macrophages. In conclusion, the present study provided evidences that THP could be served as a new immunomodulator in both functional foods and the pharmaceutical field.