Objective To prepare Flammulina velutipes crude polysaccharide (FVP) and evaluate its structural characteristics and immunomodulatory activity.Methods FVP was prepared from Flammulina velutipes using hot-water extraction and ethanol precipitation, and its structural characteristics were characterized by combining Fourier transform infrared spectroscopy (FTIR) and high-performance size-exclusion chromatography-multi-angle laser light scattering-refractive index detection (HPSEC-MALLS-RI). The immunomodulatory activity of FVP was evaluated using RAW 264.7 macrophages and a cyclophosphamide-induced immunosuppressed mouse model.Results The total sugar content of FVP is (51.39±1.36) g/100 g. Structural characterization results show that FVP has characteristic features of a β-type pyranose ring and exhibits a multi-component relative molecular weight distribution, with an overall weight-average relative molecular weight of 291.40. Further in vitro and in vivo bioactivity evaluations indicate that FVP has no obvious toxicity toward RAW 264.7 macrophages in the range of 3.125~600.0 μg/mL and can promote nitric oxide production and the mRNA expression levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6 and significantly improve the suppression of body weight gain, the decrease of lymphocyte-related indices, and the abnormalities of immune organ indices in immunosuppressed mice.Conclusion The prepared FVP has favorable immunomodulatory activity and can be applied in the field of functional foods.