Abstract: Objective: To investigate the effects of fermentation by Lactobacillus plantarum （ATCC 14917）, Saccharomyces cerevisiae （ATCC 9763）, and Bacillus subtilis （ATCC 9327） on the structural characteristics and bioactivities of mulberry polysaccharides (MPs). Methods: Three fermented MPs (MFP-Z, MFP-N, MFP-K) and an unfermented control (MFP) were prepared using ultrasound-assisted extraction. Their structural features were characterized by high-performance liquid chromatography (HPLC) and high-performance gel permeation chromatography (HPGPC). The antioxidant, hypoglycemic and immunomodulatory activities were systematically evaluated in vitro. Results: Fermentation significantly increased the yield and total sugar content of MPs, while altering their molecular weights and monosaccharide compositions. MFP, MFP-N and MFP-Z consisted of nine monosaccharides (mannose, N-acetylglucosamine, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose) with distinct molar ratios. MFP-K additionally contained galactosamine hydrochloride. Fermentation with L. plantarum increased the molecular weight of MPs, whereas B. subtilis and S. cerevisiae fermentation reduced it. FT-IR spectroscopy confirmed that the major functional groups of MPs were retained after fermentation. For bioactivities, the overall antioxidant capacity followed the order MFP-N > MFP-K > MFP-Z > MFP, and the hypoglycemic activity ranked MFP-N > MFP-Z > MFP-K > MFP. Moreover, MFP-K and MFP-N markedly enhanced macrophage phagocytosis and stimulated the secretion of NO, TNF-α, and IL-6. Conclusion: Microbial fermentation can modulate the structure and enhance the bioactivities of mulberry polysaccharides in a strain-dependent manner without damaging the main functional groups. This study provides an effective strategy for the high-value utilization of mulberry polysaccharides in functional foods and pharmaceuticals.