• A heterogalactan from ginseng flowers was purified and characterized. • It contained (1 → 4)-β-D-galactan and highly branched (1 → 6)-β-D-galactan. • The side chain of (1 → 6)-β-D-galactan was branched with α-L-Ara f through O-3. • The heterogalactan could activate macrophages in vitro and in vivo. A neutral polysaccharide fraction (WGFPN) was isolated from Panax ginseng flowers. Monosaccharide composition and HPSEC-MALLS-RI (high-performance size exclusion chromatography coupled with multi-angle laser light scattering detector and refractive index detector) analyses showed WGFPN was a heterogalactan with a molecular weight of 11.0 kDa. Methylation, 1D/2D NMR (nuclear magnetic resonance) spectra and enzymatic hydrolysis methods were used to characterize the structure of WGFPN. It possessed a less branched (1 → 4)-β-D-galactan and a significantly branched (1 → 6)-β-D-galactan. The side chains of (1 → 6)-β-D-galactan were branched with α-L-1,5-Ara f and t-α-L-Ara f residues at O-3. Trace amount of 1,4-linked Glc p , terminal Gal p , terminal Glc p and terminal Man p residues might attached to the 1,6-linked galactan through O-3 or 1,4-linked galactan through O-6 as side chains. WGFPN could activate RAW264.7 macrophages through increasing macrophage phagocytosis, releasing NO and secreting TNF-α, IL-6, IFN-γ and IL-1β in vitro. Moreover, WGFPN could enhance the immunity of cyclophosphamide (CTX)-induced immunosuppressed mice in vivo. Hence, WGFPN might be a potential natural immunomodulatory agent. [ABSTRACT FROM AUTHOR]