A soluble homogeneous β-glucan, GFPBW1, with a molecular mass of 300 kDa was purified from the fraction of the fruit bodies of Grifola frondosa extracted with 5% NaOH. Using various methods, such as infrared spectroscopy, NMR, methylation and monosaccharide composition analysis, its structure was determined to be a β-D-(1-3)-linked glucan backbone with a single β-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. It induced TNF-α and IL-6 production and the activation of Syk and NF-κB signaling in resident peritoneal macrophages from ICR mice, which could be significantly inhibited by the blocking reagent laminarin. A competitive phagocytosis assay with FITC-zymosan indicated that GFPBW1 could bind to DC-associated C-type lectin 1 (Dectin-1). The TNF-α secretion and activation of Syk/NF-κB signaling triggered by GFPBW1 were enhanced in RAW264.7 cells overexpressing wild but not mutant (Δ38 and Y15S) Dectin-1. Furthermore, GFPBW1 potentiated the Concanavalin A-induced proliferative response of splenocytes and inhibited Sarcoma-180 growth allografted in ICR mice but not in immunodeficient BALB/c nu/nu mice. These results suggested that the antitumor activity of GFPBW1 was partially associated with the activation of macrophages via the Dectin-1/Syk/NF-κB signaling pathway. This molecule could be a promising biological response modifier with clear application for antitumor therapies.