The royal sun mushroom, Agaricus brasiliensis is a widely consumed mushroom around the world. In this study, the immunoregulatory potential of A. brasiliensis polysaccharides was investigated in vitro and in vivo. In vivo, the polysaccharides remarkably increased the spleen and thymus indexes in mice, and this effect was influenced significantly by age (the adult and the juvenile). The spleen index increased by 27.28% in adult mice treated with the polysaccharides, whereas the increase in juvenile mice was just 12.59% at the dose of 150 mg·kg-1·d-1. Moreover, the effect of the polysaccharides on the thymus and spleen indexes in adult mice was obvious both in males and females. The carbon clearance ability (phagocytic index) was improved with increasing doses, (32.81% at 120 mg·kg-1·d-1, and 38.34% at 150 mg·kg-1·d-1) in mice treated with the polysaccharides. In vitro, the polysaccharides increased the RAW264.7 cell proliferation with 34.78% at 25 µg/mL and 26.78% at 50 µg/mL. Furthermore, the polysaccharides also promoted mRNA expressions of interleukin (IL)-6, IL-1β, cyclooxygenase-2, and Toll-like receptor 4 (TLR4), myeloid differentiation 88 (MYD88), and TIR-domain-containing adapter-inducing interferon-β (TRIF) in the cells, indicating that the polysaccharides induce the secretion of inflammatory cytokines by stimulating TLR4/MyD88 and TLR4/TRIF pathways. In conclusion, these results suggest that A. brasiliensis polysaccharides induce a very promising immunostimulation effect in vivo and in vitro. Therefore, it should be explored as a novel natural functional food additive.