Schisandra Sphenanthera polysaccharides fractions (SSPs), namely SSP40, SSP60, and SSP80, were obtained by gradient precipitation with 40 %, 60 %, and 80 % (v/v) ethanol, respectively. It was found that gradient ethanol precipitation (GEP) significantly affected the physicochemical and structural characteristics of SSPs, including molecular weight, monosaccharide composition, and surface morphology. Compared to fractions SSP40 and SSP60, SSP80 was observed to have a lower molecular weight (22.58 kDa) and certain specific monosaccharide composition, such as lower glucose content and higher galactose, arabinose, rhamnose, and galacturonic acid content. Furthermore, the apparent porosity of the SSPs increased with increasing ethanol concentration in GEP. After fermentation at 37 °C for 48 h, fraction SSP80 prominently promoted the production of more short-chain fatty acids (SCFAs), increasing from an initial 1.39 ± 0.08 to 26.75 ± 0.54 mmol/L. The SSP fraction types extracted by GEP greatly affected the modulation of the intestinal microflora at different levels. The SSP80 fraction with excellent structure demonstrated the best ability to modulate the intestinal microflora by increasing the relative abundance of Bacteroides, Faecalibacterium and Dialister and decreasing the relative abundance of Escherichia-Shigella. The remarkable differences in modulating the intestinal microflora confirmed the importance of carefully selecting GEP to fraction SSPs that promote health.