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• Immunostimulatory polysaccharide (EtISPFa) was purified from E. tinctorium. • EtISPFa is 1354 kDa and is a heteroglycan. • EtISPFa is a β -glucan containing high amount of glucuronic acid (10–20 %). • EtISPFa induces various cytokines production in macrophage cells. An immuno-stimulatory polysaccharide (EtISPFa) was purified from water extract of the fungus Echinodontium tinctorium. EtISPFa has an estimated weight average molecular weight (M w) of 1354 kDa and is composed of glucose (66.2 %), glucuronic acid (10.1 %), mannose (6.7 %), galactose (6.4 %), xylose (5.6 %), rhamnose (3.1 %), fucose (1.8 %), and arabinose (0.2 %). It has multiple glycosidic linkages, with 3-Glc p (19.8 %), 4-Glc p A (10.8 %), 6-Glc p (10.7 %), and 3,6-Glc p (8.7 %) being the most prominent. NMR analysis showed that EtISPFa has a backbone containing mostly of 3-substituted β -glucopyranose with 4-substituted glucopyranosyluronic acid. Short side chains consisting of an average of two β -glycopyranose residues, connected through 1→6 linkages, are attached to the 6-position of about every 4th or 5th backbone glucose residue. EtISPFa is a novel glucuronic acid-containing β -glucan capable of significantly inducing the production of cytokines IL-17, IL-16, MIP-2, G-CSF,GM-CSF, LIF, MIP-1α, MIP-1β, and RANTES in vitro. EtISPFa should be further explored for its immuno-stimulatory activity in vivo. [ABSTRACT FROM AUTHOR]