The fundamental mechanisms underlying the preventional and therapeutic effects of polysaccharides from fungi, including the immunostimulatory, antiviral and antitumor effects, are considered to occur through the modulation and stimulation of the macrophage and complement system. LDG-A, a novel polysaccharide from Lactarius deliciosus (L. ex Fr.) Gray exhibits marked antitumor activities in vivo. However, the underlying molecular mechanism of the antitumor activities of LDG-A remains unclear. In the present study, cell cycle analysis was performed in macrophages and B cells, and the transcriptomes of macrophages in the control group and LDG-A group were sequenced using Illumina sequencing technology to analyze the differentially expressed genes (DEGs), and elucidate the molecular mechanisms underlying the immunomodulatory and antitumor activities of LDG-A. The cell cycle analysis results indicated that LDG-A was able to promote the proliferation of B cells by promoting cell cycle progression in S phase and G2/M phase and eliminating cell cycle arrest in G0/G1, and promote the proliferation of macrophages by promoting cell cycle progression in G0/G1 phase and eliminating cell cycle arrest in G2/M phase. Of the total number of genes (8,140), ~77.00% were expressed [reads per kilobase per million reads (RPKM) ≥1] and 1,352 genes were highly expressed (RPKM >60) in the LDG-A group. Of 775 unigenes which were identified as DEGs, 469 were downregulated and 306 genes were upregulated. A protein chip method was also used to determine the cytokines secreted by macrophages. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and GO enrichment analysis indicated that the Janus kinase/signal transducer and activator of transcription, mitogen-activated protein kinase, chemokine, vascular endothelial growth factor and transforming growth factor β signaling pathways are markedly enriched for DEGs.