Prostate cancer is one of the most prevalent lethal diseases among men globally. In the treatment of prostate cancer, the limited therapeutic efficacy of the standard non-hormonal systemic therapy docetaxel (DTX) represents an important challenge. Cancer-associated fibroblasts (CAFs) play a crucial role in resistance to therapy because of their prevalence and functional pleiotropy in tumor environments. Our previous research revealed that MPSSS, a novel polysaccharide extracted from Lentinus edodes, could significantly attenuate the immunosuppressive function of myeloid suppressor cells and CAFs. In this study, we investigated whether MPSSS could potentiate the efficacy of DTX against prostate cancer by inhibiting CAF-induced chemoresistance and elucidated its underlying mechanisms. The sensitivity of PC-3 prostate cancer cells cultured with conditioned medium derived from CAFs (CAF-CM) to DTX was assessed. The resistance effect induced by CAF-CM was abolished when CAFs were pretreated with MPSSS. Bioinformatic analysis of datasets from the Gene Expression Omnibus database revealed the activation of the transforming growth factor β1 (TGF-β1) signaling pathway in DTX-resistant cells. Based on this finding, we demonstrated that treatment with the TGF-β1 receptor inhibitor SB525334 reversed DTX resistance in CAFs, suggesting that TGF-β1 secreted by CAFs was a crucial intermediary in the development of DTX resistance in PC3 cells. Further research revealed that MPSSS decreases the secretion of TGF-β1 by inhibiting the JAK2/STAT3 pathway via Toll-like receptor 4 in CAFs. Overall, MPSSS might be a potential adjuvant treatment for DTX resistance in prostate cancer.Keywords: Cancer-associated fibroblasts; Chemoresistance; Docetaxel; Prostate cancer; Transforming growth factor β1.