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In this study, soluble dietary fibers (SDFs) were extracted from rape bee pollen using four methods including acid extraction (AC), alkali extraction (AL), cellulase extraction (CL) and complex enzyme extraction (CE). The effects of different extraction methods on the structure of SDFs and in vitro fermentation characteristics were further investigated. The results showed that the four extraction methods significantly affected the monosaccharide composition molar ratio, molecular weight, surface microstructure and phenolic compounds content, but showed little effect on the typical functional groups and crystal structure. In addition, all SDFs decreased the Firmicutes/Bacteroidota ratio, promoted the growth of beneficial bacteria such as Bacteroides, Parabacteroides and Phascolarctobacterium, inhibited the growth of pathogenic bacteria such as Escherichia-Shigella, and increased the total short-chain fatty acids (SCFAs) concentrations by 1.63–2.45 times, suggesting that the bee pollen SDFs had a positive regulation on gut microbiota. Notably, the SDF obtained by CE exhibited the largest molecular weight, a relatively loose structure, higher extraction yield and phenolic compounds content and the highest SCFA concentration. Overall, our results indicated that CE was an appropriate extraction method of high-quality bee pollen SDF. [ABSTRACT FROM AUTHOR]