A novel polysaccharide fraction (MSCP2) was extracted and isolated from the roots of Millettia Speciosa Champ. Structural characterization revealed that MSCP2 had an average molecular weight of 2.85 × 10 Da and was composed of fucose, arabinose, galactose, glucose and xylose with a ratio of 2.20: 2.52: 4.04: 87.29: 3.96. Methylation analysis and nuclear magnetic resonance (NMR) analysis showed that the main glycosidic linkage types of MSCP2 were proved to be α-D-Glcp-(1→, →4)-α-D-Glcp-(1→, →4)-α-D-Xylp-(1→, →6)-β-D-Galp-(1→, α-L-Araf-(1→, →3,4)-β-L-Fucp-(1→ and →4)-α-D-GalpA-(1→. The immunomodulatory assay suggested that MSCP2 could significantly improve the pinocytic capacity and increase the secretion of nitric oxide (NO) and cytokines by regulating the corresponding mRNA expression in RAW 264.7 cells. The data from the membrane receptor assay demonstrated that the potential mechanisms of MSCP2-induced macrophage activation were mainly through toll-like receptor 4 (TLR4), scavenger receptor type A (SRA) and glucan receptor (GR)-mediated signaling pathways. These results suggested that MSCP2 can be developed as a promising immunomodulatory agent in functional foods.