Abstract: In order to investigate the inhibitory effect of Tricholoma matsutake polysaccharides (TMP) on UVB radiation-induced melanogenesis and its molecular mechanism, a new polysaccharide TMP-N was prepared by hot water extraction and DEAE Sepharose Fast Flow purification. The mouse melanoma cells (B16-F10) were utilised as a model to evaluate the effects of TMP-N on melanin content, antioxidant capacity and related gene expression levels after UVB irradiation. The results demonstrated that the molecular weight of TMP-N was 7819.5 Da, mainly composed of glucose, galactose, mannose and fucose. Cellular experimentation demonstrated that the treatment of TMP-N could effectively reduce melanogenesis by inhibiting tyrosinase activity and enhancing the antioxidant capacity of B16-F10 cells. The analysis of the fluorescence quantitative PCR assay demonstrated that TMP-N functioned by downregulating melanogenic pathways through modulation of both PKA and MAPK signalling cascades. This study provides theoretical support for the use of T. matsutake polysaccharides as a natural active ingredient, which is valuable for safe and efficient protection against UVB radiation damage.