To explore bioactive polysaccharides from medicinal fungi, a homogeneous heteropolysaccharide SB1-1 was extracted from the stroma of Shiraia bambusicola by enzyme assisted extraction, ethanol precipitation, anion-exchange and size-exclusion chromatographies. Chemical and spectroscopic analyses showed that SB1-1 was a neutral polysaccharide composed of mannose, glucose and galactose at a molar ratio of 1.1:1.9:1.0, and had a molecular weight of 18.3 kDa. SB1-1 was composed of a mannan core and glucose and galactose side chains. The mannan core was composed of (1 → 2)-α-Manp substituted by the side chains (1 → 6)-α-d-Manp, (1 → 4)-α-d-Glcp and (1 → 6)-α-d-Glcp with different degrees of polymerization at the C-6. The galactose side chains had the backbone of (1 → 6)-β-d-Galf. There were two branch sites on every five sugars on average at the O-2 of →6)-β-Galf(1→, and the branches consisted of →2)-β-d-Galf(1→ and terminal β-d-Galf(1→. The potential immunomodulatory activities of SB1-1 on RAW264.7 macrophages were investigated. The results showed that SB1-1 could activate macrophage and significantly improve its phagocytic ability by neutral red uptake assay. Additionally, SB1-1 enhanced the productions of IL-1, IL-6, IL-12 and TNF-α in the RAW264.7 macrophages by stimulation of SB1-1, while significantly increased the production of inducible nitric oxide synthase (iNOS) production. This study demonstrated that the galactofuranose-containing polysaccharide SB1-1 from S. bambusicola which is a novel mannogalactoglucan, could enhance the activation of macrophages.