Abstract: Objective: The aim of this study was to elucidate the structural characteristics and immunomodulatory activities of polysaccharides extracted from Sargassum fusiforme. Methods: A homogeneous fraction designated as SFP-αⅡ (uronic acid content: 95.1%) was obtained from polysaccharides that were extracted by hot water and ethanol precipitation, followed by purification through DEAE-52 cellulose and Sephadex G-200 chromatography. The structural characterization of SFP-αⅡ were investigated by high-performance gel permeation chromatography (HPGPC, homogeneity and Mw), fourier transform infrared (FT-IR, functional group), high-performance liquid chromatography (HPLC, monosaccharide composition), methylation analysis (linkage pattern), nuclear magnetic resonance (NMR, primary structure), congo red assay (triple-helix structure), circular dichroism (CD, helical conformation), atomic force microscopy (AFM, molecular morphology) and scanning electron microscopy (SEM, surface morphology). An immunosuppressed mouse model was established by intraperitoneal injection of cyclophosphamide (CTX), followed by oral administration of SFP-αⅡ. After intervention, the spleen index, thymus index, tissue structure changes, phagocytic ability of peritoneal macrophages, nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) levels were evaluated. Results: SFP-αⅡ was a homogeneous polysaccharide with a molecular weight of 16.5 kDa and had a backbone composed of →1,4)-α-GulA(1→ and →1,4)-β-ManA→ residues in a molar ratio of 33.8:66.2. SFP-αⅡ exhibited no triple-helix conformation, whereas it displayed a multi-branched or network-like helical configuration with densely interwoven aggregates of varying sizes under microscopic observation. In CTX-induced immunosuppressed mice, SFP-αⅡ administration restored body weight, spleen and thymus indices, furthermore markedly ameliorated histological damage to these organs. Moreover, SFP-αⅡ significantly enhanced macrophage phagocytic activity and NO production which elevated TNF-α and IL-1β levels in a dose-dependent manner. Conclusion: SFP-αⅡ exhibited remarkable immunoregulatory activity and effectively alleviated CTX-induced immunosuppression, which indicates its potential as a functional food ingredient for immune support.