There are ongoing efforts to explore the potential of natural bioactive substances including polysaccharides in immunological regulation and understand the mechanisms under their immune-regulating function. In this study, a polysaccharide from Flammulina velutipes (FVP-1) exhibited immunomodulatory in RAW264.7 macrophage cells and mouse spleen cells. FVP-1 increased the secretion of cytokines (like TNF-α, IL-6 and IL-1β) and their mRNA expression, upregulated the transcription and translation expression of COX-2 and iNOS, and enhanced the release of reactive oxygen species the phagocytic activity in macrophages, thereby promoting the maturation and transformation of certain lymphocytes. All these functions of FVP-1 depended to some extent on its concentration. The RSAD2 effector was involved in the immunomodulatory function of FVP-1 towards macrophages and mouse splenocytes, through mediating FVP-1's activation and regulation of the NF-κB/MAPK signaling pathway. These findings indicate the potential of FVP-1 as a natural immunomodulator and approach for improving immune function.