In this study, macrophage RAW264.7 cells were used to study the effect of Agaricus brasiliensis polysaccharide on NO release and iNOS expression, and the phosphorylation level of IκBα protein was detected to explore its mechanisms. All the studies in the present paper could provide a new theoretical basis for the development of A. brasiliensis polysaccharide. The results showed that different concentrations of the polysaccharides enhanced the NO release and the expression of iNOS protein in RAW264.7 cells in the same dose-dependent manner. In addition, A. brasiliensis polysaccharide at 25 μg/mL promoted the expression of p-IκBα protein in RAW264.7 cells, and the highest expression was in 45 min (0～60 min), demonstrating that it could activate NF-κB signal transduction pathway. In conclusion, A. brasiliensis polysaccharides enhanced the release of NO in macrophages through up-regulating the iNOS expression by NF-κB pathway.