A water-soluble polysaccharide, designated FVPA2, was isolated from the fruiting bodies of Flammulina velutipes using DEAE Sepharose Fast Flow and gel-permeation chromatography. Its structure was elucidated by monosaccharide composition and methylation analysis, ultraviolet, Fourier transform infrared spectrometry, and nuclear magnetic resonance spectroscopy. Results showed that FVPA2 was a homogeneous heteropolysaccharide containing galactose, fucose, and mannose in a molar ratio of 5:1:1. High-performance liquid chromatography indicated its molecular weight as 3.4 × 104 Da. FVPA2 also has a repeating unit. In vitro immunomodulatory studies showed that Raw264.7 cells were stimulated to secret nitric oxide upon administration of 200-500 µg/mL FVPA2. FVPA2 also stimulated the proliferation of mouse spleen lymphocytes and B lymphocytes.