Anti-tumor activity ofTremella fuciformispolysaccharides (TFPS) has been widely reported, but its mechanism remains poorly understood. In this study, we established an in vitro co-culture system (B16 melanoma cells and RAW 264.7 macrophage-like cells) to explore the potential anti-tumor mechanism of TFPS. Based on our results, TFPS exhibited no inhibition on the cell viability of B16 cells. However, significant apoptosis was observed when B16 cells were co-cultured with TFPS-treated RAW 264.7 cells. We further found that mRNA levels of M1 macrophage markers including iNOS and CD80 were significantly upregulated in TFPS-treated RAW 264.7 cells, while M2 macrophage markers such as Arg-1 and CD 206 remained unchanged. Besides, the migration, phagocytosis, production of inflammatory mediators (NO, IL-6 and TNF-α), and protein expression of iNOS and COX-2 were markedly enhanced in TFPS-treated RAW 264.7 cells. Network pharmacology analysis indicated that MAPK and NF-κB signaling pathways may be involved in M1 polarization of macrophages, and this hypothesis was verified by Western blot. In conclusion, our research demonstrated that TFPS induced apoptosis of melanoma cells by promoting M1 polarization of macrophages, and suggested TFPS may be applied as an immunomodulatory for cancer therapy.