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Shiitake mushroom (Lentinus edodes) polysaccharide (SMP), an important immunomodulatory substance, is usually hard to be absorbed by the intestinal tract, because of its macromolecule characteristics. The objective of this study was to develop the water-in-oil (W/O) nanoemulsion to improve its absorption efficiency and the biological activity. Based on the results of pseudoternary phase diagram, the formulation of nanoemulsion were optimized as IPM/polysaccharide/Tween80-Span85/anhydrous ethanol = 39/16/33.75/11.25 (v/v/v/v), used as the oil, aqueous phase, surfactant and co-surfactant, respectively. Under this condition, the droplet size of a W/O SMP nanoemulsion was 144.5 nm, with the polydispersity of 0.128 and the great stability. Moreover, the anti-tumor activity of SMP was notably improved after nano-emulsification. The results demonstrated that SMP nanoemulsion has great potential for applying in health food and pharmaceutical industry. [ABSTRACT FROM AUTHOR]