Nanotechnology has become relevant in the food-related industries, and edible mushrooms can be a potential raw material for providing satisfied edible nanomaterial. In this study, by following 3 different pretreatments (hot water or cold alkali or hot alkali) insoluble polysaccharide nanoparticles were prepared from Flammulina velutipes by wet milling and high pressure homogenization and their properties were investigated. The resultant nanoparticles were characterized by SEM, GC-MS (for its main compositions), FTIR, XRD, and TG. The 1 wt% nanoparticle dispersions presented non-Newtonian, shear-thinning fluids with the viscosity in an increasing order for the hot water < cold alkali < hot alkali. Moreover, the dynamical rheological results showed differences of storage (G') and loss (G″) moduli of these particle dispersions. It was concluded that the Flammulina velutipes-derived polysaccharides nanoparticles have great potential applications in the food industry, for example, as emulsifiers, reinforcement agents, and bioactive carriers.