•A bioactive fucomannogalactan (FMG-Hm) was isolated from H. marmoreus mushroom.•It consists of α-Galp (1→6) main chain, branched at O-2 by α-Fucp and β-Manp units.•FMG-Hm reduced malignancy parameters in melanoma cells, without toxicity.•This heteropolysaccharide exhibited promising in vitro antimelanoma effects. A fucomannogalactan (FMG-Hm), with a molecular weight of 17.1 kDa, obtained from fruiting bodies of Hypsizygus marmoreus exhibited promising in vitro antimelanoma effects. FMG-Hm was not cytotoxic, nor did it alter the cell morphology and proliferation, but was able to inhibit colony-forming ability and cell migration in B16-F10 murine melanoma cells. An analysis of the monosaccharide composition indicated that FMG-Hm was composed of fucose, mannose, and galactose in a ratio of 1.00:1.08:3.17. The FMG-Hm was structurally characterized based on methylation analysis, partial acid hydrolysis, and NMR experiments. The results indicated that FMG-Hm contained a α-(1→6)-linked galactopyranosyl main chain, partially substituted at O-2 by non-reducing ends of α-L-fucopyranose and β-D-mannopyranose. The predicted structure of the heteropolysaccharide was established as: Display omitted