Fungal polysaccharides had been approved to show manifold biological activities in the medical and health fields, including antitumor, antioxidant and immune stimulatory. The aim of this paper is to study the mechanism of the antitumor and immune-stimulatory activities of the polysaccharides from Lactarius deliciosus Gray (LDG-A) acting on macrophages. A fungal polysaccharide was fractionated and purified by using the DEAE-Cellulose 52 to fast flow column. After that the Sevag method was used for de-proteinization. The LDG-A was used to stimulate macrophages to secrete cytokines. The cytokines that were secreted by macrophages were detected by protein chips. The results indicated that 96 cytokines were up-regulated and 56 cytokines were down-regulated. All cytokines were imported into KEGG PATHWAY Database and NCBI to carry out analysis of the signalling pathway and biological function, respectively. The results showed that LDG-A could induce the immune response of macrophages through a series of signalling pathways. The major signalling pathways were JAK-STAT, PI3K-AKT and NF-kappa B. In addition, ELISA experiment was used to further verify that macrophages stimulated by LDG-A could secrete cytokines through the three major signalling pathways to regulate the antitumor and immunostimulatory activities.